Cell penetrating peptides and methods of making and using thereof

ABSTRACT

Disclosed herein are compounds having activity as cell penetrating peptides. In some examples, the compounds can comprise a cell penetrating peptide moiety and a cargo moiety. The cargo moiety can comprise one or more detectable moieties, one or more therapeutic moieties, one or more targeting moieties, or any combination thereof. In some examples, the cell penetrating peptide moiety is cyclic. In some examples, the cell penetrating peptide moiety and cargo moiety together are cyclic. In some examples, the cell penetrating peptide moiety is cyclic and the cargo moiety is appended to the cyclic cell penetrating peptide moiety structure. In some examples, the cargo moiety is cyclic and the cell penetrating peptide moiety is cyclic, and together they form a fused bicyclic system.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. patent applicationSer. No. 15/312,878, filed on Nov. 21, 2016, now U.S. Pat. No.10,626,147, which is a national phase application of InternationalPatent Application No. PCT/US2015/032043, filed May 21, 2015, whichclaims the priorities of U.S. Provisional Application 62/158,351, filedMay 7, 2015, and U.S. Provisional Application 62/001,535, filed May 21,2014, the entire contents of each of which are herein incorporated byreference in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant numbersGM062820, GM110208, and CA132855 awarded by the National Institutes ofHealth. The government has certain rights in this invention.

BACKGROUND

The plasma membrane presents a major challenge in drug discovery,especially for biologics such as peptides, proteins and nucleic acids.One potential strategy to subvert the membrane barrier and deliver thebiologics into cells is to attach them to “cell-penetrating peptides(CPPs)”. Despite three decades of investigation, the fundamental basisfor CPP activity remains elusive. CPPs that enter cells via endocytosismust exit from endocytic vesicles in order to reach the cytosol.Unfortunately, the endosomal membrane has proven to be a significantbarrier towards cytoplasmic delivery by these CPPs; often a negligiblefraction of the peptides escapes into the cell interior (El-Sayed, A etal. AAPS J., 2009, 11, 13-22; Varkouhi, A K et al. J. ControlledRelease, 2011, 151, 220-228; Appelbaum, J S et al. Chem. Biol., 2012,19, 819-830). What are thus needed are new cell penetrating peptides andcompositions comprising such peptides that can be used to deliver agentsto various cell types. The compositions and methods disclosed hereinaddress these and other needs.

SUMMARY

Disclosed herein are compounds having activity as cell penetratingpeptides. In some examples, the compounds can comprise a cellpenetrating peptide moiety and a cargo moiety. The cargo moiety cancomprise one or more detectable moieties, one or more therapeuticmoieties, one or more targeting moieties, or any combination thereof.

In some examples, the cell penetrating peptide moiety is cyclic. In someexamples, the cell penetrating peptide moiety and cargo moiety togetherare cyclic; this is referred to herein as an “endocyclic” configuration.In some examples, the cell penetrating peptide moiety is cyclic and thecargo moiety is appended to the cyclic cell penetrating peptide moietystructure; this is referred to herein as an “exocyclic” configuration.In some examples, the cargo moiety is cyclic and the cell penetratingpeptide moiety is cyclic, and together they form a fused bicyclicsystem; this is referred to herein as a “bicyclic” configuration.

In some examples, the compounds can be of Formula I:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶

AA⁷

_(m)

AA⁸

_(n)

AA⁹)_(p)   Iwherein AA¹, AA², AA³, AA⁴, AA⁵, AA⁶, AA⁷, AA⁸, and AA⁹ (i.e., AA¹-AA⁹)are each independently an amino acid; and m, n and p are independentlyselected from 0 and 1. In other examples, of Formula I, there can bemore than 9 amino acids, such that when m and p are 1, n is 2 or more.These larger peptides are disclosed with each of formula herein, e.g.,IA, II, IIa, IIb, and IIc. In some examples three or more amino acidsare arginine and one or more are phenylalanine. In still other examplesone or more amino acids is naphthylalanine or tryptophan.

In some examples, the cell penetrating peptide moiety is cyclic, and thecompounds can be of Formula Ia:

wherein AA¹-AA⁹, m, n, and p are as defined in Formula I, and whereinthe curved line indicates a covalent bond.

In some examples, the compound further comprises a cargo moiety, and thecompounds can be of Formula II:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶

AA⁷

_(m)

AA⁸

_(n)

AA⁹

_(p)car_(g)o   IIwherein the cargo moiety can comprise a detectable moiety, a therapeuticmoiety, a targeting moiety, or a combination thereof and AA¹-AA⁹, m, n,and p are as defined in Formula I.

In some examples, the cell penetrating peptide moiety and cargo moietytogether are cyclic, and the compounds are of Formula IIa:

wherein the cargo moiety is as defined in Formula II and AA¹-AA⁹, m, nand p are as defined in Formula I.

In some examples, the cell penetrating peptide moiety is cyclic and thecargo moiety is appended to the cyclic cell penetrating peptide moietystructure, and the compounds are of Formula IIb:

wherein the cargo moiety is as defined in Formula II and AA¹-AA⁹, m, nand p are as defined in Formula I.

In some examples, the cargo moiety is cyclic and the cell penetratingpeptide moiety is cyclic, and together they form a fused bicyclicsystem, and the compounds are of Formula IIc:

wherein the cargo moiety is as defined in Formula II and AA¹-AA⁹, m, nand p are as defined in Formula I.

The amino acids can be coupled by a peptide bond. The amino acids can becoupled to the cargo moiety at the amino group, the carboxylate group,or the side chain.

In some examples, at least one amino acid comprises naphthylalanine oran analogue or derivative thereof. In some examples, at least three ofthe amino acids independently comprise arginine or an analogue orderivative thereof. In some examples, at least one amino acid comprisesphenylalanine or an analogue or derivative thereof. In some examples of,at least one amino acid comprises glutamine or an analogue or derivativethereof.

In some examples, the cell penetrating peptide moeity can by any of SEQID NO:1 to SEQ ID NO:90. In some examples, the cell penetrating peptidemoiety can be a variant of any of SEQ ID NO:1 to SEQ ID NO:90.

The cargo moiety can comprise any cargo of interest, for example alinker moiety, a detectable moiety, a therapeutic moiety, a targetingmoiety, and the like, or any combination thereof.

The cargo moiety can be attached to the cell penetrating peptide moietyat the amino group, the carboxylate group, or the side chain of any ofthe amino acids of the cell penetrating peptide moiety (e.g., at theamino group, the carboxylate group, or the side chain or any ofAA¹-AA⁹).

In some examples, the therapeutic moiety comprises a targeting moiety.The targeting moiety can comprise, for example, a sequence of aminoacids that can target one or more enzyme domains. In some examples, thetargeting moiety can comprise an inhibitor against a protein that canplay a role in a disease, such as cancer, cystic fibrosis, diabetes,obesity, or combinations thereof. In some examples, the therapeuticmoiety can comprise a targeting moiety that can act as an inhibitoragainst Ras (e.g., K-Ras), PTP1B, Pin1, Grb2 SH2, CAL PDZ, and the like,or combinations thereof.

Also disclosed herein are compositions that comprise the compoundsdescribed herein. Also disclosed herein are pharmaceutically-acceptablesalts and prodrugs of the disclosed compounds.

Also provided herein are methods of use of the compounds or compositionsdescribed herein. Also provided herein are methods for treating adisease or pathology in a subject in need thereof comprisingadministering to the subject an effective amount of any of the compoundsor compositions described herein.

Also provided herein are methods of treating, preventing, orameliorating cancer in a subject. The methods include administering to asubject an effective amount of one or more of the compounds orcompositions described herein, or a pharmaceutically acceptable saltthereof. The methods of treatment or prevention of cancer describedherein can further include treatment with one or more additional agents(e.g., an anti-cancer agent or ionizing radiation).

Also described herein are methods of killing a tumor cell in a subject.The method includes contacting the tumor cell with an effective amountof a compound or composition as described herein, and optionallyincludes the step of irradiating the tumor cell with an effective amountof ionizing radiation. Additionally, methods of radiotherapy of tumorsare provided herein. The methods include contacting the tumor cell withan effective amount of a compound or composition as described herein,and irradiating the tumor with an effective amount of ionizingradiation.

In some examples of the methods of treating of treating, preventing, orameliorating cancer or a tumor in a subject, the compound or compositionadministered to the subject can comprise a therapeutic moiety that cancomprise a targeting moiety that can act as an inhibitor against Ras(e.g., K-Ras), PTP1B, Pin1, Grb2 SH2, or combinations thereof.

The disclosed subject matter also concerns methods for treating asubject having a metabolic disorder or condition. In one embodiment, aneffective amount of one or more compounds or compositions disclosedherein is administered to a subject having a metabolic disorder and whois in need of treatment thereof. In some examples, the metabolicdisorder can comprise type II diabetes. In some examples of the methodsof treating of treating, preventing, or ameliorating the metabolicdisorder in a subject, the compound or composition administered to thesubject can comprise a therapeutic moiety that can comprise a targetingmoiety that can act as an inhibitor against PTP1B.

The disclosed subject matter also concerns methods for treating asubject having an immune disorder or condition. In one embodiment, aneffective amount of one or more compounds or compositions disclosedherein is administered to a subject having an immune disorder and who isin need of treatment thereof. In some examples of the methods oftreating of treating, preventing, or ameliorating the immune disorder ina subject, the compound or composition administered to the subject cancomprise a therapeutic moiety that can comprise a targeting moiety thatcan act as an inhibitor against Pin1.

The disclosed subject matter also concerns methods for treating asubject having cystic fibrosis. In one embodiment, an effective amountof one or more compounds or compositions disclosed herein isadministered to a subject having cystic fibrosis and who is in need oftreatment thereof. In some examples of the methods of treating thecystic fibrosis in a subject, the compound or composition administeredto the subject can comprise a therapeutic moiety that can comprise atargeting moiety that can act as an inhibitor against CAL PDZ.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF FIGURES

The accompanying Figures, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIGS. 1A, 1B, and 1C display structures showing cargo attachment duringendocyclic (A), exocyclic (B), and bicyclic (C) delivery of cargos(shown in light grey) by cFΦR₄.

FIG. 2 displays the structures of some of the peptides used in thisstudy.

FIG. 3 displays a scheme showing the synthesis of cFΦR₄-S-S-GFP.

FIG. 4 displays a scheme showing the synthesis of cFΦR₄-PTP1B.

FIGS. 5A and 5B display the binding of FITC-labeled cFΦR₄, R₉ and Tat to(FIG. 5A) SUV and (FIG. 5B) heparin sulfate.

FIGS. 6A and 6B display representative live-cell confocal images ofHEK293 cells treated for 2 h with rhodamine B-labeled peptides andfluid-phase uptake marker, dextran^(FITC). (FIG. 6A) Cells treated with5 μM cFΦR₄-A₅ and dextran^(FITC) in the same Z-section. (FIG. 6B) Cellstreated with 5 μM cFΦR₄-R₅ and dextran^(FITC) in the same Z-section.

FIG. 7 displays the effect of cFΦR₄ on the endocytosis ofdextran^(Alexa488) by HeLa cells. HeLa cells were treated with clearDMEM containing no supplement, 1 μM cFΦR₄ only, 100 μMdextran^(Alexa488) only, or both 1 μM cFΦR₄ and 100 μMdextran^(Alexa488). MFI, mean fluorescence intensity.

FIG. 8 displays the effect of pH on CAP fluorescence. cFΦR₄-PCP wasdephosphorylated by alkaline phosphatase and purified by HPLC and itsfluorescence at indicated pH's was measured.

FIGS. 9A, 9B, and 9C display the internalization of pCAP-containingpeptides into cultured cells: I, untagged PCP; II, cFΦR₄-PCP; III,cFΦR₄-PCP and Na₃VO₄; IV, R₉-PCP; V, Tat-PCP; and VI, Antp-PCP. (FIG.9A) Representative live-cell confocal images of HEK293 cells treatedwith 5 μM peptides. Top panel, nuclear stain with DRAQS; bottom panel,CAP fluorescence in the same Z-section. (FIG. 9B) Flow cytometry of HeLacells treated with 0 or 10 μM peptides. (FIG. 9C) CAP fluorescence from(FIG. 9B) after subtraction of background fluorescence (untreatedcells). MFI, mean fluorescence intensity.

FIGS. 10A, 10B, 10C, 10D, 10E, and 10F displays representative live-cellconfocal microscopic images of HEK293 cells treated for 2 h withrhodamine B-labeled peptides (5 μM each) and fluid-phase endocytosismarker, dextran^(FITC) (0.5 mg/mL). The red fluorescence of rhodamine Band the green fluorescence of dextran^(FITC) from the same Z-section andtheir merged image are shown in each panel. The enlarged images of atypical cell(s) are shown in each case in order to show theintracellular distribution of the internalized peptides. (FIG. 10A)Cells treated with bicyclo(FΦR₄-A₅)^(Rho); (FIG. 10B)monocyclo(FΦR₄-A₅)^(Rho); (FIG. 10C) bicyclo(FΦR₄-A₇)^(Rho); (FIG. 10D)monocyclo(FΦR₄-A₇)^(Rho); (FIG. 10E) bicyclo(FΦR₄—RARAR)^(Rho); and(FIG. 10F) bicyclo(FΦR₄-DADAD)^(Rho).

FIGS. 11A and 11B display: (FIG. 11A) Structures of CPP-S-S-GFPconjugates; and (FIG. 11B) Live-cell confocal images of mammalian cellsafter 2-h treatment with 1 μM GFP (I), Tat-S-S-GFP (II), orcFΦR₄-S-S-GFP (III) and nuclear stain DRAQS. All images were recorded inthe same Z-section.

FIGS. 12A and 12B display: (FIG. 12A) Western blot analysis of theglobal pY protein levels of NIH 3T3 cells after treatment with 0-500 nMPTP1B or cFΦR₄-PTP1B (IB: anti-pY antibody 4G10); and (FIG. 12B) Samesamples as in (FIG. 12A) were analyzed by SDS-PAGE and coomassie bluestaining. M, molecular-weight markers.

FIGS. 13A and 13B display: (FIG. 13A) Comparison of the serum stabilityof cFΦR₄, Tat, R₉, and Antp; and (FIG. 13B) Cytotoxicity of cFΦR₄. Theindicated cell lines were treated with DMSO (control), 5 μM, or 50 μMcFΦR₄ for 24 h and the percentage of live cells was determined by MTTassay.

FIGS. 14A and 14B display MTT assay of various mammalian cells aftertreatment with cFΦR₄ (5 or 50 μM) for (FIG. 14A) 48 h or (FIG. 14B) 72h.

FIG. 15 displays a diagram showing the points along the endocyticpathway where cFΦR₄, R₉, and Tat escape into the cytoplasm and wherespecific inhibitors are proposed to function.

FIG. 16 displays scheme showing the reversible cyclization strategy fordelivering linear peptidyl cargos into mammalian cells. GSH,glutathione.

FIGS. 17A, 17B, 17C, 17D, 17E, and 17F display: (FIG. 17A) Synthesis ofdisulfide-bond cyclized peptide. (FIG. 17B) Synthesis of thioether-bondcyclized peptide. Reagents and conditions: (a) Standard Fmoc/HATUchemistry; (b) piperidine/DMF; (c) 3,3′-dithiodipropionic acid/DIC; (d)β-mercaptoethanol/DMF; (e) modified reagent K; (f) trituration; (g)DMSO/DPBS (pH 7.4). (h) 4-bromobutyric acid/DIC; (i) 1% TFA/DCM; (j) 1%DIPEA/DMF; PG, protecting group. Trt, trityl; Mmt, methoxytrityl. (FIG.17C) Structures of FITC labeled peptides 1 and 2. (FIG. 17D) Structuresof pCAP (phosphocoumaryl aminopropionic acid) containing peptides 1-PCPand 2-PCP. (FIG. 17E) Structures of Amc (7-amino-4-methylcourmarin)containing caspase fluorogenic substrates 3-7. (FIG. 17F) Structures ofFITC labeled CAL-PDZ domain ligands 9-11.

FIGS. 18A and 18B display (FIG. 18A) Live-cell confocal microscopicimages of HeLa cells treated with 5 μM FITC-labeled peptide 1 (I) or 2(II), endocytosis marker Dextran^(Rho) (0.5 mg mL⁻¹), and nuclear stainDRAQS. Images in different fluorescence channels were all recorded inthe same Z-section. (FIG. 18B) Flow cytometry of HeLa cells treated with5 μM FITC-labeled peptides 1, 2, or FITC alone.

FIGS. 19A and 19B display: (FIG. 19A) FACS analysis of HeLa cellstreated with 0 or 5 μM peptides 1-PCP, 2-PCP for 2 h; and (FIG. 19B) CAPfluorescence from (FIG. 19A) after subtraction of backgroundfluorescence (untreated cell). MFI, mean fluorescence intensity.

FIG. 20 displays a comparison of the proteolytic stability of peptides 1and 2.

FIG. 21 displays the time-dependent release of fluorogenic coumarinproduct by Jurkat cells treated with peptides 3-7 (5 μM) in the absenceand presence of 100 μM caspase inhibitor Z-VAD(OMe)-FMK (FMK).

FIGS. 22A, 22B, 22C, 22D, and 22E display: (FIG. 22A) Structure ofCAL-PDZ inhibitor 8. (FIG. 22B) Binding of peptide 8 to CAL-PDZ domainin the presence or absence of reducing reagent. (FIG. 22C) Live-cellmicroscopic images of HeLa cells treated with peptide 8 (5 μM) and DRAQSin the same Z-section. I, green fluorescence of internalized peptide 8;II, overlay of green peptide fluorescence and blue nuclear stain. (FIG.22D) Immunofluorescent staining showing the distribution of CFTR in thepresence or absence of Corr-4a (10 μM) and unlabeled peptide 8 (50 μM).(FIG. 22E) SPQ assays showing CFTR-specific stimulation-inducedfluorescence increase in slope in the absence or presence of VX809 (20μM) and peptide 8 (50 μM). P values were calculated from two-tailedt-test.

FIG. 23 displays a schematic of the evolution of a cell-permeable PTP1Binhibitor.

FIG. 24 displays a schematic of the design and synthesis of cyclicpeptide library. Reagents and conditions: (a) standard Fmoc/HBTUchemistry; (b) soak in water; (c) 0.1 equiv Fmoc-Glu(δ-NHS)-OAll, 0.4equiv Boc-Met-OH in Et₂O/CH₂Cl₂; (d) piperidine; (e) split into twoparts; (f) split-and-pool synthesis by Fmoc/HATU chemistry; (g)Pd(PPh₃)₄; (h) PyBOP, HOBt; and (i) Reagent K. X², 10% F₂Pmp and 90%Tyr; X¹ and X³—X⁵, random positions; Φ, L-2-naphthylalanine; CPP,cell-penetrating motif FΦR₄ or R₄ΦF.

FIGS. 25A and 25B display the competitive inhibition of PTP1B bymonocyclic peptide inhibitor 2. (FIG. 25A) Lineweaver-Burk plots forPTP1B-catalyzed hydrolysis of pNPP (0-24 mM) in the presence of varyingconcentrations of inhibitor 2 (0, 22.5, 45, and 90 nM). (FIG. 25B)Secondary plot of the Michaelis constant ratio (K/K₀) as a function of.

FIGS. 26A, 26B, and 26C display: (FIG. 26A) live-cell confocalmicroscopic images (same Z-section) of A549 lung cancer cells aftertreatment for 2 h with 5 μM FITC-labeled inhibitor 2 (top panel) or 4(bottom panel) and endocytosis marker dextran^(Rho) (1.0 mg/mL); (FIG.26B) Lineweaver-Burk plot showing competitive inhibition of PTP1B by 0,28, 56, and 112 nM inhibitor 4; and (FIG. 26C) Sensitivity of variousPTPs to inhibition by inhibitor 4 (all activities were relative to thatin the absence of inhibitor).

FIG. 27 displays the solid-phase synthesis of inhibitor 4. Reagents andconditions: a) standard Fmoc chemistry; b) trimesic acid, HBTU; c)Pd(PPh₃)₄, N-methylaniline; d) PyBOP; e) TFA.

FIG. 28 displays a comparison of the serum stability of monocyclic PTP1Binhibitor 2 and bicyclic inhibitor 4.

FIGS. 29A, 29B, 29C, and 29D display: (FIG. 29A) Global pY proteinlevels in A549 cells after treatment with 0-5 μM inhibitor 4 for 2 h;(FIG. 29B) SDS-PAGE analysis (Coomassie blue staining) of the samesamples from (FIG. 29A) shows uniform sample loading in all lanes; (FIG.29C) Effect of inhibitor 4 on insulin receptor phosphorylation atTyr¹¹⁶² and Tyr¹¹⁶³ sites. HepG2 cells were treated with indicatedconcentrations of inhibitor 4 for 2 h and then stimulated with insulin(100 nM) for 5 min, followed by SDS-PAGE and immunoblotting withanti-IRpY¹¹⁶²/pY¹¹⁶³ antibody; and (FIG. 29D) Quantitation of IR pYlevels from (FIG. 29C) (data shown are the mean±SD from five independentexperiments).

FIG. 30 displays the conversion of impermeable Pin1 inhibitor into acell-permeable bicyclic inhibitor.

FIGS. 31A, 31B, 31C, 31D, and 31E display the FA analysis of the bindingof Pin1 inhibitor 5-9 to Pin1, respectively.

FIGS. 32A and 32B display the competition for binding to Pin1 byinhibitors 5 and 7. Each reaction contained 0.1 μM FITC-labeledinhibitor 5, 1 μM Pin1, and 0-5 μM unlabeled inhibitor 5 (FIG. 3A) orinhibitor 7 (FIG. 32B) and the FA value was measured and plotted againstthe competitor concentration.

FIGS. 33A, 33B, and 33C display the cellular uptake of Pin1 inhibitors.Live-cell confocal microscopic images of HEK293 cells treated with 5 μMFITC-labeled Pin1 inhibitor 5 A(FIG. 33A) or 7 (FIG. 33B) and 1 mg/mLendocytosis marker Dextran^(Rho) for 2 h. All images were recorded atthe same Z-section. (FIG. 33C) FACS analysis of HeLa cells after 2-htreatment with DMSO or 5 μM FITC-labeled Pin1 inhibitor 5, 7, 8, or 9.MFI, mean fluorescence intensity. Procedure: Hela cells were cultured insix-well plates (2×10⁵ cells per well) for 24 h. On the day ofexperiment, the cells were incubated with 5 μM FITC labeled bicyclicpeptide or control monocylic peptide in phenol red-free DMEMsupplemented with 1% FBS. After 2 h, the peptide solution was removed,and the cells were washed with DPBS, treated with 0.25% trypsin for 5min, washed again with DPBS. Finally, the cells were resuspended in theflow cytometry buffer and analyzed by flow cytometry (BD FACS Aria),with excitation at 535 nm.

FIG. 34 displays the effect of Pin1 Inhibitors 5, 7, 8, and 9 on cancercell proliferation. HeLa cells (100 μL/each well, 5×10⁴ cells/mL) wereseeded in a 96-well culture plate and allowed to grow overnight in DMEMsupplemented with 10% FBS. Varying concentrations of Pin1 inhibitor (0-5μM) were added to the wells and the cells were incubated at 37° C. with5% CO₂ for 72 h. After that, 10 μL of a MTT stock solution (5 mg/mL) wasadded into each well. The plate was incubated at 37° C. for 4 h and 100μL of SDS-HCl solubilizing solution was added into each well, followedby thorough mixing. The plate was incubated at 37° C. overnight and theabsorbance of the formazan product was measured at 570 nm on a MolecularDevices Spectramax M5 plate reader. Each experiment was performed intriplicates and the cells untreated with peptide were used as control.

FIGS. 35A, 35B, 35C, and 35D display live cell confocal images of mouseventricular cardiac myocytes after treatment for 3 h with 5 μMc(FΦRRRRQ)-K(FITC) (FIG. 35A) and c(fΦRrRrQ)-K(FITC) (FIG. 35B). FIG.35C displays labeling of calmodulin (T5C) with cyclic cell penetratingpeptide through a disulfide bond. FIG. 35D displays live cell confocalimages of mouse ventricular cardiac myocytes after treatment for 3 hwith 6 μM cFΦR4-conjugated Cy3-labeled calmodulin.

FIG. 36 displays the evolution of bicyclic peptide inhibitors againstPin1. The structural moieties derived from library screening are shownin grey, while the changes made during optimization are shown in lightgrey.

FIGS. 37A, 37B, 37C, and 37D displays the characterization of peptide37. (FIG. 37A) Binding to FITC-labeled peptide 37 to Pin1 as analyzed byfluorescent anisotropy (FA). (FIG. 37B) Competition between peptide 37and FITC-labeled peptide 1 (100 nM) for binding to Pin1 (400 nM) asmonitored by FA. (FIG. 37C) Effect of peptide 37 on the cis-transisomerase activity of Pin1, Pin4, FKBP12, and cyclophilin A usingSuc-Ala-Glu-Pro-Phe-pNA as substrate. (FIG. 37D) Comparison of the serumstability of peptides 1 and 37.

FIGS. 38A, 38B, 38C, and 38D display cellular activity of peptide 37.(FIG. 38A) Cellular uptake of peptides 1, 37, and 46 (5 μM) by HeLacells as analyzed by flow cytometry. MFI, mean fluorescence intensity;none, untreated cells (no peptide). (FIG. 38B) Anti-proliferative effectof peptides 37, 46, and 47 on HeLa cells as measured by MTT assay. (FIG.38C) Western blots showing the effect of peptides 1, 37 and 47 on theprotein level of PML in HeLa cells. β-Actin was used as loading control.(FIG. 38D) Quantification of western blot results from (FIG. 38C). Datareported were after background subtraction and represent the mean±SDfrom 3 independent experiments.

DETAILED DESCRIPTION

The compounds, compositions, and methods described herein may beunderstood more readily by reference to the following detaileddescription of specific aspects of the disclosed subject matter and theExamples and Figures included therein.

Before the present compounds, compositions, and methods are disclosedand described, it is to be understood that the aspects described beloware not limited to specific synthetic methods or specific reagents, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

Also, throughout this specification, various publications arereferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference into this application in order tomore fully describe the state of the art to which the disclosed matterpertains. The references disclosed are also individually andspecifically incorporated by reference herein for the material containedin them that is discussed in the sentence in which the reference isrelied upon.

General Definitions

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined to have the followingmeanings.

Throughout the description and claims of this specification the word“comprise” and other forms of the word, such as “comprising” and“comprises,” means including but not limited to, and is not intended toexclude, for example, other additives, components, integers, or steps.

As used in the description and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a composition”includes mixtures of two or more such compositions, reference to “anagent” includes mixtures of two or more such agents, reference to “thecomponent” includes mixtures of two or more such components, and thelike.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. By “about” is meant within5% of the value, e.g., within 4, 3, 2, or 1% of the value. When such arange is expressed, another aspect includes from the one particularvalue and/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another aspect. It will befurther understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

As used herein, by a “subject” is meant an individual. Thus, the“subject” can include domesticated animals (e.g., cats, dogs, etc.),livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratoryanimals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds.“Subject” can also include a mammal, such as a primate or a human. Thus,the subject can be a human or veterinary patient. The term “patient”refers to a subject under the treatment of a clinician, e.g., physician.

The term “inhibit” refers to a decrease in an activity, response,condition, disease, or other biological parameter. This can include butis not limited to the complete ablation of the activity, response,condition, or disease. This can also include, for example, a 10%reduction in the activity, response, condition, or disease as comparedto the native or control level. Thus, the reduction can be a 10, 20, 30,40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between ascompared to native or control levels.

By “reduce” or other forms of the word, such as “reducing” or“reduction,” is meant lowering of an event or characteristic (e.g.,tumor growth). It is understood that this is typically in relation tosome standard or expected value, in other words it is relative, but thatit is not always necessary for the standard or relative value to bereferred to. For example, “reduces tumor growth” means reducing the rateof growth of a tumor relative to a standard or a control.

By “prevent” or other forms of the word, such as “preventing” or“prevention,” is meant to stop a particular event or characteristic, tostabilize or delay the development or progression of a particular eventor characteristic, or to minimize the chances that a particular event orcharacteristic will occur. Prevent does not require comparison to acontrol as it is typically more absolute than, for example, reduce. Asused herein, something could be reduced but not prevented, but somethingthat is reduced could also be prevented. Likewise, something could beprevented but not reduced, but something that is prevented could also bereduced. It is understood that where reduce or prevent are used, unlessspecifically indicated otherwise, the use of the other word is alsoexpressly disclosed. For example, the terms “prevent” or “suppress” canrefer to a treatment that forestalls or slows the onset of a disease orcondition or reduced the severity of the disease or condition. Thus, ifa treatment can treat a disease in a subject having symptoms of thedisease, it can also prevent or suppress that disease in a subject whohas yet to suffer some or all of the symptoms.

The term “treatment” refers to the medical management of a patient withthe intent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

The term “anticancer” refers to the ability to treat or control cellularproliferation and/or tumor growth at any concentration.

The term “therapeutically effective” refers to the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses or symptoms of a disease or disorder. Such amelioration onlyrequires a reduction or alteration, not necessarily elimination.

The term “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problems or complications commensurate witha reasonable benefit/risk ratio.

The term “carrier” means a compound, composition, substance, orstructure that, when in combination with a compound or composition, aidsor facilitates preparation, storage, administration, delivery,effectiveness, selectivity, or any other feature of the compound orcomposition for its intended use or purpose. For example, a carrier canbe selected to minimize any degradation of the active ingredient and tominimize any adverse side effects in the subject.

The terms “peptide,” “protein,” and “polypeptide” are usedinterchangeably to refer to a natural or synthetic molecule comprisingtwo or more amino acids linked by the carboxyl group of one amino acidto the alpha amino group of another.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer, diastereomer, and meso compound,and a mixture of isomers, such as a racemic or scalemic mixture.

Reference will now be made in detail to specific aspects of thedisclosed materials, compounds, compositions, articles, and methods,examples of which are illustrated in the accompanying Examples andFigures.

Compounds

Disclosed herein are compounds having activity as cell penetratingpeptides. In some examples, the compounds can comprise a cellpenetrating peptide moiety and a cargo moiety. The cargo moiety cancomprise one or more detectable moieties, one or more therapeuticmoieties, one or more targeting moieties, or any combination thereof.

In some examples, the cell penetrating peptide moiety is cyclic. In someexamples, the cell penetrating peptide moiety and cargo moiety togetherare cyclic. In some examples, the cell penetrating peptide moiety iscyclic and the cargo moiety is appended to the cyclic cell penetratingpeptide moiety structure. In some examples, the cargo moiety is cyclicand the cell penetrating peptide moiety is cyclic, and together theyform a fused bicyclic system.

The cell penetrating peptide moiety can comprise five or more, morespecifically six or more, for example, six to twelve, or six to nineamino acids. When there are six to nine amino acids the compounds can beof Formula I:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶

AA⁷

_(m)

AA⁸

_(n)

AA⁹)_(p)   Iwherein AA¹, AA², AA³, AA⁴, AA⁵, AA⁶, AA⁷, AA⁸, and AA⁹ (i.e., AA¹-AA⁹)are each independently an amino acid; and m, n and p are independentlyselected from 0 and 1. Wherein there are more than 9 amino acids,Formula I can have m and p each be 1 and n can be 2 or more, e.g., 2 to10 or 2 to 5. In some examples three or more amino acids are arginineand one or more are phenylalanine. In still other examples one or moreamino acids is naphthylalanine or tryptophan.

In some examples, the compounds can be of Formula I:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶

AA⁷

_(m)

AA⁸

_(n)

AA⁹)_(p)   Iwherein AA¹, AA², AA³, AA⁴, AA⁵, AA⁶, AA⁷, AA⁸, and AA⁹ (i.e., AA¹-AA⁹)are each independently an amino acid; and m, n and p are independentlyselected from 0 and 1.

In some examples, the cell penetrating peptide moiety is cyclic, and thecompounds can be of Formula Ia:

wherein AA¹-AA⁹, m, n, and p are as defined in Formula I, and whereinthe curved line indicates a covalent bond. The curved line can be acovalent bond in the backbone of the peptide (i.e., the carboxylic acidof one AA forming an amide bond with the α-amine of another AA), a bondbetween the side chains of two AAs, a bond from one side chain of an AAto either the backbone carboxylic acid or α-amine of another AA, or adisulfide bond between two AAs.

In some examples, the compound further comprises a cargo moiety, and thecompounds can be of Formula II:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶

AA⁷

_(m)

AA⁸

_(n)

AA⁹

_(p)car_(g)o   IIwherein the cargo moiety can comprise a detectable moiety, a therapeuticmoiety, a targeting moiety, or a combination thereof and AA¹-AA⁹, m, n,and p are as defined in Formula I.

In some examples, the cell penetrating peptide moiety and cargo moietytogether are cyclic, and the compounds are of Formula IIa:

wherein the cargo moiety is as defined in Formula II and AA¹-AA⁹, m, nand p are as defined in Formula I.

In some examples, the cell penetrating peptide moiety is cyclic and thecargo moiety is appended to the cyclic cell penetrating peptide moietystructure, and the compounds are of Formula IIb:

wherein the cargo moiety is as defined in Formula II and AA¹-AA⁹, m, nand p are as defined in Formula I.

In some examples, the cargo moiety is cyclic and the cell penetratingpeptide moiety is cyclic, and together they form a fused bicyclicsystem, and the compounds are of Formula IIc:

wherein the cargo moiety is as defined in Formula II and AA¹-AA⁹, m, nand p are as defined in Formula I.

Cell Penetrating Peptide

The cell penetrating peptide moeity comprises at least 5, morespecifically, at least 6 amino acids, even more specifically from from 6to 12, from 6 to 9, from 6 to 7, from 7 to 8, from 8 to 9, and morespecifically 6, 7, 8, or 9 amino acids. For the endocyclic motif, atleast 5 amino acids can be used. It is also disclosed herein that forthe endocyclic structure, some amino acids in the penetrating peptidemoiety can also be part of the cargo moiety. For example, a peptidepenetrating moiety FNalRR can be formed when from FNal and an cargomoiety with two Args. In this case, the two Arg residues perform dualfunctions. Thus, in some cases the sequence of the cargo moiety is takeninto account when referring to the peptide penetrating moiety.

For the exocyclic motif, at least 6 amino acids can be used with, forexample, glutamine being used to attach the cargo.

Each amino acid can be a natural or non-natural amino acid. The term“non-natural amino acid” refers to an organic compound that is acongener of a natural amino acid in that it has a structure similar to anatural amino acid so that it mimics the structure and reactivity of anatural amino acid. The non-natural amino acid can be a modified aminoacid, and/or amino acid analog, that is not one of the 20 commonnaturally occurring amino acids or the rare natural amino acidsselenocysteine or pyrolysine. Non-natural amino acids can also be theD-isomer of the natural amino acids. Examples of suitable amino acidsinclude, but are not limited to, alanine, alloisoleucine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, naphthylalanine,phenylalanine, proline, pyroglutamic acid, serine, threonine,tryptophan, tyrosine, valine, a derivative, or combinations thereof.These, and others, are listed in the Table 1 along with theirabbreviations used herein.

TABLE 1 Amino Acid Abbreviations Amino Acid Abbreviations* alanine Ala(A) allosoleucine AIle arginine Arg (R) asparagine Asn (N) aspartic acidAsp (D) cysteine Cys (C) cyclohexylalanine Cha 2,3-diaminopropionic acidDap 4-fluorophenylalanine Fpa (Σ) glutamic acid Glu (E) glutamine Gln(Q) glycine Gly (G) histidine His (H) homoproline Pip (Θ) isoleucine Ile(I) leucine Leu (L) lysine Lys (K) methionine Met (M) napthylalanine Nal(Φ) norleucine Nle (Ω) phenylalanine Phe (F) phenylglycine Phg (Ψ)4-(phosphonodifluoromethyl)phenylalanine F₂Pmp (Λ) pipecolic acid Pp ( 

 ) proline Pro (P) sarcosine Sar (Ξ) selenocysteine Sec (U) serine Ser(S) threonine Thr (T) tyrosine Tyr (Y) tryptophan Trp (W) valine Val (V)*single letter abbreviations: when shown incapital letters herein itindicates the L-amino acid form, when shown in lower case herein itindicates the D-amino acid form

The amino acids can be coupled by a peptide bond. The amino acids can becoupled to the cargo moiety at the amino group, the carboxylate group,or the side chain.

In some examples of Formula I, at least one amino acid comprisesnaphthylalanine or tryptophan, or analogues or derivatives thereof. Insome examples of Formula I, at least three of the amino acidsindependently comprise arginine or an analogue or derivative thereof. Insome examples of Formula I, at least one amino acid comprisesphenylalanine, phenylglycine, or histidine, or analogues or derivativesthereof. In some examples of Formula I, at least one amino acidcomprises glutamine or an analogue or derivative thereof.

In some examples, the cell penetrating peptide (CPP) moiety can be anyof the sequences listed in Table 2. In some examples, the cellpenetrating peptide can be the reverse of any of the sequences listed inTable 2. In some examples, the cell penetrating peptide sequence can bea cyclic form of any of the sequences listed in Table 2.

TABLE 2 CPP sequences - linear or cyclic SEQ ID NO CPP sequence #AA's #Rresidues 1 FΦRRRQ 6 3 2 FΦRRRC 6 3 3 FΦRRRU 6 3 4 RRRΦFQ 6 3 5 RRRRΦF 64 6 FΦRRRR 6 4 7 FϕRrRq 7 3 8 FϕRrRQ 7 3 9 FΦRRRRQ 7 4 10 fΦRrRrQ 7 4 11RRFRΦRQ 7 4 12 FRRRRΦQ 7 4 13 rRFRΦRQ 7 4 14 RRΦFRRQ 7 4 15 CRRRRFWQ 7 416 FfΦRrRrQ 8 4 17 FFΦRRRRQ 8 4 18 RFRFRΦRQ 8 4 19 URRRRFWQ 8 4 20CRRRRFWQ 8 4 21 FΦRRRRQK 8 4 22 FΦRRRRQC 8 4 23 fΦRrRrRQ 8 5 24 FΦRRRRRQ8 5 25 RRRRΦFDΩC 9 4 26 FΦRRR 5 3 27 FWRRR 5 3 28 RRRΦF 5 3 29 RRRWF 5 3Φ = L-naphthylalanine; ϕ = D-naphthylalanine; Ω = L-norleucine

Certain embodiments of the invention include amino acid sequenceswherein at least four consecutive amino acids have alternatingchirality. As used herein, chirality refers to the “D” and “L” isomersof amino acids. In particular embodiments of the invention, at leastfour consecutive amino acids have alternating chirality and theremaining amino acids are L-amino acids. In other embodiments, thepeptides of the invention comprise a four amino acid sequence havingD-L-D-L chirality. In still other embodiments, the peptides of theinvention comprise a four amino acid sequence having L-D-L-D chirality.

In embodiments, peptides of the invention comprise two consecutiveL-amino acids. In further embodiments, peptides of the inventioncomprise two consecutive L-amino acids separating two D-amino acids. Inyet further embodiments, peptides of the invention comprise twoconsecutive L-amino acids separating two D-amino acids and at least fourconsecutive amino acids having alternating chirality, such as, but notlimited to peptide sequences with D-L-L-D-L-D or L-D-L-L-D-L-Dchirality. In even further embodiments, peptides of the inventioncomprise two consecutive L-amino acids separating two D-amino acids andat least five consecutive amino acid having alternating chirality, suchas, but not limited to peptide sequences with D-L-L-D-L-D-L orL-D-L-L-D-L-D-L chirality.

In embodiments, peptides of the invention comprise two consecutiveD-amino acids. In further embodiments, peptides of the inventioncomprise two consecutive D-amino acids separating two L-amino acids. Instill further embodiments of the invention, peptides of the inventioncomprise two consecutive D-amino acids separating two L-amino acids andat least four consecutive amino acids having alternating chirality, suchas, but not limited to peptide sequences with L-D-D-L-D-L. In evenfurther embodiments of the invention, peptides of the invention comprisetwo consecutive D-amino acids separating two L-amino acids and at leastfive consecutive amino acids having alternating chirality, such as, butnot limited to peptide sequences with L-D-D-L-D-L-D.

In some embodiments, the amino acid sequence with alternating chiralitycomprises about at least about 4 amino acids, at least about 5 aminoacids, at least about 6 amino acids, at least about 7 amino acids, atleast about 8 amino acids or at least about 9 amino acids. Inembodiments, the amino acid sequence with alternating chiralitycomprises of from about 4 amino acids to about 9 amino acids, or about 5amino acids to about 6 amino acids, or about 7 amino acids to about 9amino acids, or about 8 amino acids to about 9 amino acids, or about 4amino acids to about 8 amino acids, or about 4 amino acids to about 7amino acids, or about 4 amino acids to about 6 amino acids, or about 4amino acids to about 5 amino acids.

In particular embodiments, the cyclic cell-penetrating peptides of theinvention demonstrate improved cellular uptake efficiency as compared toc(FΦRRRRQ) (290-1F).

As used herein cellular uptake efficiency refers to the ability of acyclic peptide sequence to traverse a cell membrane. In embodiments,cellular uptake of the cyclic, cell penetrating peptide is not dependenton a receptor or a cell type.

In particular embodiments, uptake efficiency is determined by comparing(i) the amount of a cyclic cell-penetrating peptide of the inventioninternalized by a cell type (e.g., HeLa cells) to (ii) the amount ofc(FΦRRRRQ) (290-1F) internalized by the same cell type. To measurecellular uptake efficiency, the cell type may be incubated in thepresence of a cell-penetrating peptide of the invention for a specifiedperiod of time (e.g., 30 minutes, 1 hour, 2 hours, etc.) after which theamount of the cell-penetrating peptide internalized by the cell isquantified. Separately, the same concentration of c(FΦRRRRQ) (290-1F) isincubated in the presence of the cell type over the same period of time,and the amount of the second peptide internalized by the cell isquantified. Quantification can be achieved by fluorescently labeling thecell-penetrating peptide (e.g., with a FTIC dye) and measuring thefluorescence intensity using techniques well-known in the art.

In certain embodiments, peptides of the invention comprising at leastfour consecutive amino acid having alternating chirality have an uptakeefficiency that is superior to that of a second cyclic peptide whereinthe second cyclic peptide has an otherwise identical amino acid sequenceconsisting of L-amino acids. In some embodiments, uptake efficiency canbe improved by at least about 1.5 fold, at least about 2 fold, at leastabout 2.5 fold, at least about 3 fold, at least about 3.5 fold, at leastabout 4 fold, at least about 4.5 fold, at least about 5 fold, at leastabout 5.5 fold, at least about 6 fold, at least about 6.5 fold, at leastabout 7 fold, at least about 7.5 fold, at least about 8 fold, at leastabout 8.5 fold, at least about 9 fold, at 9.5 fold, or at least about 10fold. In other embodiments, the uptake efficiency can be improved withinthe range of from about 1.5 fold to about 10 fold, or about 2 fold toabout 10 fold, or about 2 fold to about 9.5 fold, or about 2 fold toabout 9 fold, or about 2 fold to about 8.5 fold, or about 2 fold toabout 8 fold, or about 2 fold to about 7.5 fold, or about 2 fold toabout 7 fold, or about 2 fold to about 6.5 fold, or about 2 fold toabout 6 fold, or about 2.5 fold to about 7 fold, or about 3 fold toabout 7 fold, or about 3.5 fold to about 7 fold, or about 4 to about 7,or about 4.5 fold to about 7 fold, or about 5 fold to about 7 fold, orabout 5.5 fold to about 7 fold, or about 6 fold to about 7 fold.

In certain embodiments, peptides of the invention comprising at leastfour consecutive amino acid having alternating chirality have a superioruptake efficiency as compared to c(FΦRRRRQ) (290-1F). In someembodiments, uptake efficiency can be improved by at least about 1.5fold, at least about 2 fold, at least about 2.5 fold, at least about 3fold, at least about 3.5 fold, at least about 4 fold, at least about 4.5fold, at least about 5 fold, at least about 5.5 fold, at least about 6fold, at least about 6.5 fold, at least about 7 fold, at least about 7.5fold, at least about 8 fold, at least about 8.5 fold, at least about 9fold, at 9.5 fold, or at least about 10 fold. In other embodiments, theuptake efficiency can be improved within the range of from about 1.5fold to about 10 fold, or about 2 fold to about 10 fold, or about 2 foldto about 9.5 fold, or about 2 fold to about 9 fold, or about 2 fold toabout 8.5 fold, or about 2 fold to about 8 fold, or about 2 fold toabout 7.5 fold, or about 2 fold to about 7 fold, or about 2 fold toabout 6.5 fold, or about 2 fold to about 6 fold, or about 2.5 fold toabout 7 fold, or about 3 fold to about 7 fold, or about 3.5 fold toabout 7 fold, or about 4 to about 7, or about 4.5 fold to about 7 fold,or about 5 fold to about 7 fold, or about 5.5 fold to about 7 fold, orabout 6 fold to about 7 fold.

In certain embodiments, the peptides of the invention comprise at leastone hydrophobic residue. In further embodiments, the peptides of theinvention comprise two hydrophobic residues. In still furtherembodiments, the peptides of the invention comprise at least twohydrophobic residues. In certain embodiments, at least one hydrophobicresidue is an aromatic hydrophobic residue. In particular embodiments,at least one hydrophobic residue is selected from the group consistingof naphthylalanine, phenylalanine, tryptophan, and tyrosine. In furtherembodiments, at least one hydrophobic residue is selected from the groupconsisting of naphthylalanine and phenylalanine. In certain embodiments,peptides of the invention comprise at least one naphthylalanine. In yetother embodiments, peptides of the invention comprise at least onephenylalanine. In still other embodiments, peptides of the inventioncomprise at least one phenylalanine and at least one naphthylalanine. Incertain embodiments of the invention, the peptide comprises at least onehydrophobic residue in the AA¹, AA², or AA³ position. In certainembodiments of the invention, the peptide comprises at least onearomatic hydrophobic residue in the AA¹, AA², or AA³ position. Infurther embodiments of the invention, the peptide comprises at least onehydrophobic residue selected from the group consisting ofnaphthylalanine and phenylalanine in the AA¹, AA², or AA³ position.

In some examples, the cell penetrating peptide moeity can by any of SEQID NO:1 to SEQ ID NO:29. In some examples, the cell penetrating peptidemoiety can be a variant of any of SEQ ID NO:1 to SEQ ID NO:29. Peptidevariants are well understood to those of skill in the art and caninvolve amino acid sequence modifications. For example, amino acidsequence modifications typically fall into one or more of three classes:substitutional, insertional, or deletional variants. Insertions includeamino and/or carboxyl terminal fusions as well as intrasequenceinsertions of single or multiple amino acid residues. Insertionsordinarily will be smaller insertions than those of amino or carboxylterminal fusions, for example, on the order of 1 to 3 residues.Deletions are characterized by the removal of one or more amino acidresidues from the peptide sequence. Typically, no more than from 1 to 3residues are deleted at any one site within the peptide. Amino acidsubstitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 3 amino acid residues; and deletions will rangeabout from 1 to 3 residues. Deletions or insertions preferably are madein adjacent pairs, i.e. a deletion of 2 residues or insertion of 2residues. Substitutions, deletions, insertions or any combinationthereof can be combined to arrive at a final construct. Substitutionalvariants are those in which at least one residue has been removed and adifferent residue inserted in its place. Such substitutions generallyare made in accordance with the following Table 3 and are referred to asconservative substitutions.

TABLE 3 Amino Acid Substitutions Exemplary Conservative SubstitutionsAla replaced by ser Leu replaced by ile or val Arg replaced by lys orgln Lys replaced by arg or gln Asn replaced by gln or his Met replacedby leu or ile Asp replaced by glu Phe replaced by met, leu, tyr, or fpaCys replaced by ser Ser replaced by thr Gln replaced by asn or lys Thrreplaced by ser Glu replaced by asp Trp replaced by tyr Gly replaced bypro Tyr replaced by trp or phe His replaced by asn or gln Val replacedby ile or leu Ile replaced by leu or val Nal replaced by Trp or Phe

Substantial changes in function are made by selecting substitutions thatare less conservative than those in Table 3, i.e., selecting residuesthat differ more significantly in their effect on maintaining (a) thestructure of the peptide backbone in the area of the substitution, forexample as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site or (c) the bulk of theside chain. The substitutions which in general are expected to producethe greatest changes in the protein properties will be those in which(a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for(or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl,valyl or alanyl; (b) a cysteine or proline is substituted for (or by)any other residue; (c) a residue having an electropositive side chain,e.g., lysyl, argininyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g., glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having a side chain, e.g., glycine, in this case, (e) byincreasing the number of sites for sulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the peptidesprovided herein.

It is understood that one way to define the variants of the disclosedcell penetrating peptide moieties is through defining the variants interms of homology/identity to specific known sequences. For example, SEQID NO:1 to SEQ ID NO:29 each sets forth a particular sequence.Specifically disclosed are variants of these peptide that have at least,85%, 90%, 95%, or 97% homology to SEQ ID NO:1 to SEQ ID NO:29. Those ofskill in the art readily understand how to determine the homology of twoproteins. For example, the homology can be calculated after aligning thetwo sequences so that the homology is at its highest level.

In addition to variants of SEQ ID NO:1 to SEQ ID NO:29 are derivativesof these peptides which also function in the disclosed methods andcompositions. Derivatives are formed by replacing one or more residueswith a modified residue, where the side chain of the residue has beenmodified. Additional examples are shown in Tables 6 and 18 and includevariants thereof.

Cargo Moiety

The cargo moiety can comprise any cargo of interest, for example alinker moiety, a detectable moiety, a therapeutic moiety, a targetingmoiety, and the like, or any combination thereof. In some examples, thecargo moiety can comprise one or more additional amino acids (e.g., K,UK, TRV); a linker (e.g., bifunctional linker LC-SMCC); coenzyme A;phosphocoumaryl amino propionic acid (pCAP); 8-amino-3,6-dioxaoctanoicacid (miniPEG); L-2,3-diaminopropionic acid (Dap or J);L-β-naphthylalanine; L-pipecolic acid (Pip); sarcosine; trimesic acid;7-amino-4-methylcourmarin (Amc); fluorescein isothiocyanate (FITC);L-2-naphthylalanine; norleucine; 2-aminobutyric acid; Rhodamine B (Rho);Dexamethasone (DEX); or combinations thereof.

In some examples the cargo moiety can comprise any of those listed inTable 4, or derivatives or combinations thereof.

TABLE 4 Example cargo moieties SEQ ID NO Abbreviation Sequence* 30 R₅RRRRR 31 A₅ AAAAA 32 F₄ FFFF 33 PCP DE(pCAP)LI 34 A₇ AAAAAAA 35 RARAR 36DADAD 37 DΩUD 38 UTRV *pCAP, phosphocoumaryl amino propionic acid; Ω,norleucine; U, 2-aminobutyric acid.

Detectable Moiety

The detectable moiety can comprise any detectable label. Examples ofsuitable detectable labels include, but are not limited to, a UV-Vislabel, a near-infrared label, a luminescent group, a phosphorescentgroup, a magnetic spin resonance label, a photosensitizer, aphotocleavable moiety, a chelating center, a heavy atom, a radioactiveisotope, a isotope detectable spin resonance label, a paramagneticmoiety, a chromophore, or any combination thereof. In some embodiments,the label is detectable without the addition of further reagents.

In some embodiments, the detectable moiety is a biocompatible detectablemoiety, such that the compounds can be suitable for use in a variety ofbiological applications. “Biocompatible” and “biologically compatible”,as used herein, generally refer to compounds that are, along with anymetabolites or degradation products thereof, generally non-toxic tocells and tissues, and which do not cause any significant adverseeffects to cells and tissues when cells and tissues are incubated (e.g.,cultured) in their presence.

The detectable moiety can contain a luminophore such as a fluorescentlabel or near-infrared label. Examples of suitable luminophores include,but are not limited to, metal porphyrins; benzoporphyrins;azabenzoporphyrine; napthoporphyrin; phthalocyanine; polycyclic aromatichydrocarbons such as perylene, perylene diimine, pyrenes; azo dyes;xanthene dyes; boron dipyoromethene, aza-boron dipyoromethene, cyaninedyes, metal-ligand complex such as bipyridine, bipyridyls,phenanthroline, coumarin, and acetylacetonates of ruthenium and iridium;acridine, oxazine derivatives such as benzophenoxazine; aza-annulene,squaraine; 8-hydroxyquinoline, polymethines, luminescent producingnanoparticle, such as quantum dots, nanocrystals; carbostyril; terbiumcomplex; inorganic phosphor; ionophore such as crown ethers affiliatedor derivatized dyes; or combinations thereof. Specific examples ofsuitable luminophores include, but are not limited to, Pd (II)octaethylporphyrin; Pt (II)-octaethylporphyrin; Pd (II)tetraphenylporphyrin; Pt (II) tetraphenylporphyrin; Pd (II)meso-tetraphenylporphyrin tetrabenzoporphine; Pt (II) meso-tetraphenymetrylbenzoporphyrin; Pd (II) octaethylporphyrin ketone; Pt (II)octaethylporphyrin ketone; Pd (II)meso-tetra(pentafluorophenyl)porphyrin; Pt (II) meso-tetra(pentafluorophenyl) porphyrin; Ru (II)tris(4,7-diphenyl-1,10-phenanthroline) (Ru (dpp)₃); Ru (II)tris(1,10-phenanthroline) (Ru(phen)₃), tris(2,2′-bipyridine)rutheniurn(II) chloride hexahydrate (Ru(bpy)₃); erythrosine B; fluorescein;fluorescein isothiocyanate (FITC); eosin; iridium (III)((N-methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin)); indium (III)((benzothiazol-2-yl)-7-(diethylamino)-coumarin))-2-(acetylacetonate);Lumogen dyes; Macroflex fluorescent red; Macrolex fluorescent yellow;Texas Red; rhodamine B; rhodamine 6G; sulfur rhodamine; m-cresol; thymolblue; xylenol blue; cresol red; chlorophenol blue; bromocresol green;bromcresol red; bromothymol blue; Cy2; a Cy3; a Cy5; a Cy5.5; Cy7;4-nitirophenol; alizarin; phenolphthalein; o-cresolphthalein;chlorophenol red; calmagite; bromo-xylenol; phenol red; neutral red;nitrazine; 3,4,5,6-tetrabromphenolphtalein; congo red; fluorescein;eosin; 2′,7′-dichlorofluorescein; 5(6)-carboxy-fluorecsein;carboxynaphthofluorescein; 8-hydroxypyrene-1,3,6-trisulfonic acid;semi-naphthorhodafluor; semi-naphthofluorescein; tris(4,7-diphenyl-1,10-phenanthroline) ruthenium (II) dichloride;(4,7-diphenyl-1,10-phenanthroline) ruthenium (II) tetraphenylboron;platinum (II) octaethylporphyin; dialkylcarbocyanine;dioctadecylcycloxacarbocyanine; fluorenylmethyloxycarbonyl chloride;7-amino-4-methylcourmarin (Amc); green fluorescent protein (GFP); andderivatives or combinations thereof.

In some examples, the detectable moiety can comprise Rhodamine B (Rho),fluorescein isothiocyanate (FITC), 7-amino-4-methylcourmarin (Amc),green fluorescent protein (GFP), or derivatives or combinations thereof.

The detectable moiety can be attached to the cell penetrating peptidemoiety at the amino group, the carboxylate group, or the side chain ofany of the amino acids of the cell penetrating peptide moiety (e.g., atthe amino group, the carboxylate group, or the side chain or any ofAA¹-AA^(x)).

Therapeutic Moiety

The disclosed compounds can also comprise a therapeutic moiety. In someexamples, the cargo moiety comprises a therapeutic moiety. Thedetectable moiety can be linked to a therapeutic moiety or thedetectable moiety can also serve as the therapeutic moiety. Therapeuticmoiety refers to a group that when administered to a subject will reduceone or more symptoms of a disease or disorder.

The therapeutic moiety can comprise a wide variety of drugs, includingantagonists, for example enzyme inhibitors, and agonists, for example atranscription factor which results in an increase in the expression of adesirable gene product (although as will be appreciated by those in theart, antagonistic transcription factors can also be used), are allincluded. In addition, therapeutic moiety includes those agents capableof direct toxicity and/or capable of inducing toxicity towards healthyand/or unhealthy cells in the body. Also, the therapeutic moiety can becapable of inducing and/or priming the immune system against potentialpathogens.

The therapeutic moiety can, for example, comprise an anticancer agentantiviral agent, antimicrobial agent, anti-inflammatory agent,immunosuppressive agent, anesthetics, or any combination thereof.

The therapeutic moiety can comprise an anticancer agent. Exampleanticancer agents include 13-cis-Retinoic Acid,2-Amino-6-Mercaptopurine, 2-CdA, 2-Chlorodeoxyadenosine, 5-fluorouracil,6-Thioguanine, 6-Mercaptopurine, Accutane, Actinomycin-D, Adriamycin,Adrucil, Agrylin, Ala-Cort, Aldesleukin, Alemtuzumab, Alitretinoin,Alkaban-AQ, Alkeran, All-transretinoic acid, Alpha interferon,Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide,Anandron, Anastrozole, Arabinosylcytosine, Aranesp, Aredia, Arimidex,Aromasin, Arsenic trioxide, Asparaginase, ATRA, Avastin, BCG, BCNU,Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin,Bortezomib, Busulfan, Busulfex, C225, Calcium Leucovorin, Campath,Camptosar, Camptothecin-11, Capecitabine, Carac, Carboplatin,Carmustine, Carmustine wafer, Casodex, CCNU, CDDP, CeeNU, Cerubidine,cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine,Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine,Cytarabine liposomal, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin,Darbepoetin alfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride,Daunorubicin liposomal, DaunoXome, Decadron, Delta-Cortef, Deltasone,Denileukin diftitox, DepoCyt, Dexamethasone, Dexamethasone acetate,Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC,Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin liposomal, Droxia,DTIC, DTIC-Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar,Emcyt, Epirubicin, Epoetin alfa, Erbitux, Erwinia L-asparaginase,Estramustine, Ethyol, Etopophos, Etoposide, Etoposide phosphate,Eulexin, Evista, Exemestane, Fareston, Faslodex, Femara, Filgrastim,Floxuridine, Fludara, Fludarabine, Fluoroplex, Fluorouracil,Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR,Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin,Gemzar, Gleevec, Lupron, Lupron Depot, Matulane, Maxidex,Mechlorethamine, -Mechlorethamine Hydrochlorine, Medralone, Medrol,Megace, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna,Mesnex, Methotrexate, Methotrexate Sodium, Methylprednisolone, Mylocel,Letrozole, Neosar, Neulasta, Neumega, Neupogen, Nilandron, Nilutamide,Nitrogen Mustard, Novaldex, Novantrone, Octreotide, Octreotide acetate,Oncospar, Oncovin, Ontak, Onxal, Oprevelkin, Orapred, Orasone,Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred,PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON,PEG-L-asparaginase, Phenylalanine Mustard, Platinol, Platinol-AQ,Prednisolone, Prednisone, Prelone, Procarbazine, PROCRIT, Proleukin,Prolifeprospan 20 with Carmustine implant, Purinethol, Raloxifene,Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon alfa-2a), Rubex,Rubidomycin hydrochloride, Sandostatin, Sandostatin LAR, Sargramostim,Solu-Cortef, Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin,Taxol, Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide,Thalomid, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide,Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab,Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid,Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs,Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP-16, Vumon,Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid, Zometa,Gliadel wafer, Glivec, GM-CSF, Goserelin, granulocyte colony stimulatingfactor, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine,HMM, Hycamtin, Hydrea, Hydrocort Acetate, Hydrocortisone, Hydrocortisonesodium phosphate, Hydrocortisone sodium succinate, Hydrocortonephosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin,Idarubicin, Ifex, IFN-alpha, Ifosfamide, IL 2, IL-11, Imatinib mesylate,Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEGconjugate), Interleukin 2, Interleukin-11, Intron A (interferonalfa-2b), Leucovorin, Leukeran, Leukine, Leuprolide, Leurocristine,Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine, L-PAM, L-Sarcolysin,Meticorten, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol, MTC, MTX,Mustargen, Mustine, Mutamycin, Myleran, Iressa, Irinotecan,Isotretinoin, Kidrolase, Lanacort, L-asparaginase, and LCR. Thetherapeutic moiety can also comprise a biopharmaceutical such as, forexample, an antibody.

In some examples, the therapeutic moiety can comprise an antiviralagent, such as ganciclovir, azidothymidine (AZT), lamivudine (3TC), etc.

In some examples, the therapeutic moiety can comprise an antibacterialagent, such as acedapsone; acetosulfone sodium; alamecin; alexidine;amdinocillin; amdinocillin pivoxil; amicycline; amifloxacin; amifloxacinmesylate; amikacin; amikacin sulfate; aminosalicylic acid;aminosalicylate sodium; amoxicillin; amphomycin; ampicillin; ampicillinsodium; apalcillin sodium; apramycin; aspartocin; astromicin sulfate;avilamycin; avoparcin; azithromycin; azlocillin; azlocillin sodium;bacampicillin hydrochloride; bacitracin; bacitracin methylenedisalicylate; bacitracin zinc; bambermycins; benzoylpas calcium;berythromycin; betamicin sulfate; biapenem; biniramycin; biphenaminehydrochloride; bispyrithione magsulfex; butikacin; butirosin sulfate;capreomycin sulfate; carbadox; carbenicillin disodium; carbenicillinindanyl sodium; carbenicillin phenyl sodium; carbenicillin potassium;carumonam sodium; cefaclor; cefadroxil; cefamandole; cefamandole nafate;cefamandole sodium; cefaparole; cefatrizine; cefazaflur sodium;cefazolin; cefazolin sodium; cefbuperazone; cefdinir; cefepime; cefepimehydrochloride; cefetecol; cefixime; cefmenoxime hydrochloride;cefmetazole; cefmetazole sodium; cefonicid monosodium; cefonicid sodium;cefoperazone sodium; ceforanide; cefotaxime sodium; cefotetan; cefotetandisodium; cefotiam hydrochloride; cefoxitin; cefoxitin sodium;cefpimizole; cefpimizole sodium; cefpiramide; cefpiramide sodium;cefpirome sulfate; cefpodoxime proxetil; cefprozil; cefroxadine;cefsulodin sodium; ceftazidime; ceftibuten; ceftizoxime sodium;ceftriaxone sodium; cefuroxime; cefuroxime axetil; cefuroxime pivoxetil;cefuroxime sodium; cephacetrile sodium; cephalexin; cephalexinhydrochloride; cephaloglycin; cephaloridine; cephalothin sodium;cephapirin sodium; cephradine; cetocycline hydrochloride; cetophenicol;chloramphenicol; chloramphenicol palmitate; chloramphenicol pantothenatecomplex; chloramphenicol sodium succinate; chlorhexidine phosphanilate;chloroxylenol; chlortetracycline bisulfate; chlortetracyclinehydrochloride; cinoxacin; ciprofloxacin; ciprofloxacin hydrochloride;cirolemycin; clarithromycin; clinafloxacin hydrochloride; clindamycin;clindamycin hydrochloride; clindamycin palmitate hydrochloride;clindamycin phosphate; clofazimine; cloxacillin benzathine; cloxacillinsodium; cloxyquin; colistimethate sodium; colistin sulfate; coumermycin;coumermycin sodium; cyclacillin; cycloserine; dalfopristin; dapsone;daptomycin; demeclocycline; demeclocycline hydrochloride; demecycline;denofungin; diaveridine; dicloxacillin; dicloxacillin sodium;dihydrostreptomycin sulfate; dipyrithione; dirithromycin; doxycycline;doxycycline calcium; doxycycline fosfatex; doxycycline hyclate; droxacinsodium; enoxacin; epicillin; epitetracycline hydrochloride;erythromycin; erythromycin acistrate; erythromycin estolate;erythromycin ethylsuccinate; erythromycin gluceptate; erythromycinlactobionate; erythromycin propionate; erythromycin stearate; ethambutolhydrochloride; ethionamide; fleroxacin; floxacillin; fludalanine;flumequine; fosfomycin; fosfomycin tromethamine; fumoxicillin;furazolium chloride; furazolium tartrate; fusidate sodium; fusidic acid;gentamicin sulfate; gloximonam; gramicidin; haloprogin; hetacillin;hetacillin potassium; hexedine; ibafloxacin; imipenem; isoconazole;isepamicin; isoniazid; josamycin; kanamycin sulfate; kitasamycin;levofuraltadone; levopropylcillin potassium; lexithromycin; lincomycin;lincomycin hydrochloride; lomefloxacin; Lomefloxacin hydrochloride;lomefloxacin mesylate; loracarbef; mafenide; meclocycline; meclocyclinesulfosalicylate; megalomicin potassium phosphate; mequidox; meropenem;methacycline; methacycline hydrochloride; methenamine; methenaminehippurate; methenamine mandelate; methicillin sodium; metioprim;metronidazole hydrochloride; metronidazole phosphate; mezlocillin;mezlocillin sodium; minocycline; minocycline hydrochloride; mirincamycinhydrochloride; monensin; monensin sodiumr; nafcillin sodium; nalidixatesodium; nalidixic acid; natainycin; nebramycin; neomycin palmitate;neomycin sulfate; neomycin undecylenate; netilmicin sulfate;neutramycin; nifuiradene; nifuraldezone; nifuratel; nifuratrone;nifurdazil; nifurimide; nifiupirinol; nifurquinazol; nifurthiazole;nitrocycline; nitrofurantoin; nitromide; norfloxacin; novobiocin sodium;ofloxacin; onnetoprim; oxacillin; oxacillin sodium; oximonam; oximonamsodium; oxolinic acid; oxytetracycline; oxytetracycline calcium;oxytetracycline hydrochloride; paldimycin; parachlorophenol; paulomycin;pefloxacin; pefloxacin mesylate; penamecillin; penicillin G benzathine;penicillin G potassium; penicillin G procaine; penicillin G sodium;penicillin V; penicillin V benzathine; penicillin V hydrabamine;penicillin V potassium; pentizidone sodium; phenyl aminosalicylate;piperacillin sodium; pirbenicillin sodium; piridicillin sodium;pirlimycin hydrochloride; pivampicillin hydrochloride; pivampicillinpamoate; pivampicillin probenate; polymyxin B sulfate; porfiromycin;propikacin; pyrazinamide; pyrithione zinc; quindecamine acetate;quinupristin; racephenicol; ramoplanin; ranimycin; relomycin;repromicin; rifabutin; rifametane; rifamexil; rifamide; rifampin;rifapentine; rifaximin; rolitetracycline; rolitetracycline nitrate;rosaramicin; rosaramicin butyrate; rosaramicin propionate; rosaramicinsodium phosphate; rosaramicin stearate; rosoxacin; roxarsone;roxithromycin; sancycline; sanfetrinem sodium; sarmoxicillin;sarpicillin; scopafungin; sisomicin; sisomicin sulfate; sparfloxacin;spectinomycin hydrochloride; spiramycin; stallimycin hydrochloride;steffimycin; streptomycin sulfate; streptonicozid; sulfabenz;sulfabenzamide; sulfacetamide; sulfacetamide sodium; sulfacytine;sulfadiazine; sulfadiazine sodium; sulfadoxine; sulfalene;sulfamerazine; sulfameter; sulfamethazine; sulfamethizole;sulfamethoxazole; sulfamonomethoxine; sulfamoxole; sulfanilate zinc;sulfanitran; sulfasalazine; sulfasomizole; sulfathiazole; sulfazamet;sulfisoxazole; sulfisoxazole acetyl; sulfisboxazole diolamine;sulfomyxin; sulopenem; sultamricillin; suncillin sodium; talampicillinhydrochloride; teicoplanin; temafloxacin hydrochloride; temocillin;tetracycline; tetracycline hydrochloride; tetracycline phosphatecomplex; tetroxoprim; thiamphenicol; thiphencillin potassium;ticarcillin cresyl sodium; ticarcillin disodium; ticarcillin monosodium;ticlatone; tiodonium chloride; tobramycin; tobramycin sulfate;tosufloxacin; trimethoprim; trimethoprim sulfate; trisulfapyrimidines;troleandomycin; trospectomycin sulfate; tyrothricin; vancomycin;vancomycin hydrochloride; virginiamycin; or zorbamycin.

In some examples, the therapeutic moiety can comprise ananti-inflammatory agent.

In some examples, the therapeutic moiety can comprise dexamethasone(Dex).

In other examples, the therapeutic moiety comprises a therapeuticprotein. For example, some people have defects in certain enzymes (e.g.,lysosomal storage disease). It is disclosed herein to deliver suchenzymes/proteins to human cells by linking to the enzyme/protein to oneof the disclosed cell penetrating peptides. The disclosed cellpenetrating peptides have been tested with proteins (e.g., GFP, PTP1B,actin, calmodulin, troponin C) and shown to work.

In some examples, the therapeutic moiety comprises a targeting moiety.The targeting moiety can comprise, for example, a sequence of aminoacids that can target one or more enzyme domains. In some examples, thetargeting moiety can comprise an inhibitor against an enzyme that canplay a role in a disease, such as cancer, cystic fibrosis, diabetes,obesity, or combinations thereof. For example, the targeting moiety cancomprise any of the sequences listed in Table 5.

TABLE 5 Example targeting moieties SEQ ID NO Abbreviation* Sequence 39PΘGΛYR Pro-Pip-Gly-F₂Pmp-Tyr- 40 SΘIΛΛR Ser-Pip-Ile-F₂Pmp-F₂Pmp- 41IHIΛIR Ile-His-Ile-F₂Pmp-Ile- 42 AaIΛΘR Ala-(D-Ala)-Ile-F₂Pmp-Pip- 43ΣSΘΛvR Fpa-Ser-Pip-F₂Pmp-(D-Val)- 44 ΘnPΛAR Pip-(D-Asn)-Pro-F₂Pmp-Ala-45 TΨAΛGR Tyr-Phg-Ala-F₂Pmp-Gly- 46 AHIΛaR Ala-His-Ile-F₂Pmp-(D-Ala)- 47GnGΛpR Gly-(D-Asn)-Gly-F₂Pmp-(D-Pro)- 48 fQΘΛIR(D-Phe)-Gln-Pip-F₂Pmp-Ile- 49 SPGΛHR Ser-Pro-Gly-F₂Pmp-His- 50 ΘYIΛHRPip-Tyr-Ile-F₂Pmp-His- 51 SvPΛHR Ser-(D-Val)-Pro-F₂Pmp-His- 52 AIPΛnRAla-Ile-Pro-F₂Pmp-(D-Asn)- 53 ΣSIΛQF Fpa-Ser-Ile-F₂Pmp-Gln- 54 AaΨΛfRAla-(D-Ala)-Phg-F₂Pmp-(D-Phe)- 55 ntΨΛΨR (D-Asn)-(D-Thr)-Phg-F₂Pmp-Phg-56 IPΨΛΩR Ile-Pro-Phg-F₂Pmp-Nle- 57 QΘΣΛΘR Gln-Pip-Fpa-F₂Pmp-Pip- 58nAΣΛGR (D-Asn)-Ala-Fpa-F₂Pmp-Gly- 59 ntYΛAR(D-Asn)-(D-Thr)-Tyr-F₂Pmp-Ala- 60 eAΨΛvR (D-Glu)-Ala-Phg-F₂Pmp-(D-Val)-61 IvΨΛAR Ile-(D-Val)-Phg-F₂Pmp-Ala- 62 YtΨΛARTyr-(D-Thr)-Phg-F₂Pmp-Ala- 63 nΘΨΛIR (D-Asn)-Pip-Phg-F₂Pmp-Ile- 64ΘnWΛHR Pip-(D-Asn)-Trp-F₂Pmp-His- 65 YΘvΛIR Tyr-Pip-(D-Val)-F₂Pmp-Ile-66 nSAΛGR (D-Asn)-Ser-(D-Ala)-F₂Pmp-Gly- 67 tnvΛaR(D-Thr)-(D-Asn)-(D-Val)-F₂Pmp-(D-Ala)- 68 ntvΛtR(D-Asn)-(D-Thr)-(D-Val)-F₂Pmp-(D-Thr)- 69 SItΛYRSer-Ile-(D-Thr)-F₂Pmp-Tyr- 70 nΣnΛlR (D-Asn)-Fpa-(D-Asn)-F₂Pmp-(D-Leu)-71 YnnΛΩR Tyr-(D-Asn)-(D-Asn)-F₂Pmp-Nle- 72 nYnΛGR(D-Asn)-Tyr-(D-Asn)-F₂Pmp-Gly- 73 AWnΛAR Ala-Trp-(D-Asn)-F₂Pmp-Ala- 74vtHΛYR (D-Val)-(D-Thr)-His-F₂Pmp-Tyr- 75 PΨHΛΘR Pro-Phg-His-F₂Pmp-Pip-76 nΨHΛGR (D-Asn)-Phg-His-F₂Pmp-Gly- 77 PAHΛGR Pro-Ala-His-F₂Pmp-Gly- 78AYHΛIR Ala-Tyr-His-F₂Pmp-Ile- 79 nΘeΛYR (D-Asn)-Pip-(D-Glu)-F₂Pmp-Tyr-80 vSSΛtR (D-Val)-Ser-Ser-F₂Pmp-(D-Thr)- 81 aΞt′  

 Φ′YNK ((D-Ala)-Sar-(D-pThr)-Pp-Nal-Tyr-Gln)-Lys 82 Tm(aΞt′ 

 Φ′RA)Dap Tm((D-Ala)-Sar-(D-pThr)-Pp-Nal-Arg-Ala)-Dap 83 Tm(aΞt′ 

 Φ′RAa)Dap Tm((D-Ala)-Sar-(D-pThr)-Pp-Nal-Arg-Ala-(D- Ala))-Dap 84Tm(aΞt 

 Φ′RAa)Dap Tm((D-Ala)-Sar-(D-Thr)-Pp-Nal-Arg-Ala-(D- Ala))-Dap 85Tm(aΞtaΦ′RAa)Dap Tm((D-Ala)-Sar-(D-Thr)-(D-Ala)-Nal-Arg-Ala-(D-Ala))-Dap *Fpa, Σ: L-4-fluorophenylalanine; Pip, Θ: L-homoproline;Nle, Ω: L-norleucine; Phg, Ψ L-phenylglycine; F₂Pmp, Λ:L-4-(phosphonodifluoromethyl)phenylalanine; Dap, L-2,3-diaminopropionicacid; Nal, Φ′: L-β-naphthylalanine; Pp, 

 : L-pipecolic acid; Sar, Ξ: sarcosine; Tm, trimesic acid.

The targeting moitiey and cell penetrating peptide moiety can overlap,that is residues that form the cell penetrating peptide moiety can alsobe part of the sequence that forms the targeting moiety, and vice aversa.

The therapeutic moiety can be attached to the cell penetrating peptidemoiety at the amino group, the carboxylate group, or the side chain ofany of the amino acids of the cell penetrating peptide moiety (e.g., atthe amino group, the carboxylate group, or the side chain or any ofAA¹-AA^(x)). In some examples, the therapeutic moiety can be attached tothe detectable moiety.

In some examples, the therapeutic moiety can comprise a targeting moietythat can act as an inhibitor against Ras (e.g., K-Ras), PTP1B, Pin1,Grb2 SH2, CAL PDZ, and the like, or combinations thereof.

Ras is a protein that in humans is encoded by the RAS gene. The normalRas protein performs an essential function in normal tissue signaling,and the mutation of a Ras gene is implicated in the development of manycancers. Ras can act as a molecular on/off switch, once it is turned onRas recruits and activates proteins necessary for the propagation ofgrowth factor and other receptors' signal. Mutated forms of Ras havebeen implicated in various cancers, including lung cancer, colon cancer,pancreatic cancer, and various leukemias.

Protein-tyrosine phosphatase 1B (PTP1B) is a prototypical member of thePTP superfamily and plays numerous roles during eukaryotic cellsignaling. PTP1B is a negative regulator of the insulin signalingpathway, and is considered a promising potential therapeutic target, inparticular for the treatment of type II diabetes. PIP1B has also beenimplicated in the development of breast cancer.

Pin1 is an enzyme that binds to a subset of proteins and plays a role asa post phosphorylation control in regulating protein function. Pin1activity can regulate the outcome of proline-directed kinase signalingand consequently can regulate cell proliferation and cell survival.Deregulation of Pin1 can play a role in various diseases. Theup-regulation of Pin1 may be implicated in certain cancers, and thedown-regulation of Pin1 may be implicated in Alzheimer's disease.Inhibitors of Pin1 can have therapeutic implications for cancer andimmune disorders.

Grb2 is an adaptor protein involved in signal transduction and cellcommunication. The Grb2 protein contains one SH2 domain, which can bindtyrosine phosphorylated sequences. Grb2 is widely expressed and isessential for multiple cellular functions. Inhibition of Grb2 functioncan impair developmental processes and can block transformation andproliferation of various cell types.

It was recently reported that the activity of cystic fibrosis membraneconductance regulator (CFTR), a chloride ion channel protein mutated incystic fibrosis (CF) patients, is negatively regulated byCFTR-associated ligand (CAL) through its PDZ domain (CAL-PDZ) (Wolde, Met al. J. Biol. Chem. 2007, 282, 8099). Inhibition of the CFTR/CAL-PDZinteraction was shown to improve the activity of ΔPhe508-CFTR, the mostcommon form of CFTR mutation (Cheng, S H et al. Cell 1990, 63, 827;Kerem, B S et al. Science 1989, 245, 1073), by reducing itsproteasome-mediated degradation (Cushing, P R et al. Angew. Chem. Int.Ed. 2010, 49, 9907). Thus, disclosed herein is a method for treating asubject having cystic fibrosis by administering an effective amount of acompound or composition disclosed herein. The compound or compositionadministered to the subject can comprise a therapeutic moiety that cancomprise a targeting moiety that can act as an inhibitor against CALPDZ. Also, the dcompositions or compositions disclosed herein can beadministered with a molecule that corrects the CFTR function.

Specific Examples

In some examples, the compounds can be of Formula I:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶

AA⁷

_(m)

AA⁸

_(n)

AA⁹)_(p)   Iwherein AA¹, AA², AA³, AA⁴, AA⁵, AA⁶, AA⁷, AA⁸, and AA⁹ (i.e., AA¹-AA⁹)are each independently an amino acid; and m, n and p are independentlyselected from 0 and 1.

In some examples of Formula I, m, n, and p are 0 and the compounds areof Formula I-1:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶   I-1wherein AA¹-AA⁶ are as defined in Formula I.

In some examples of Formula I, m is 1, and n and p are 0, and thecompounds are of Formula I-2:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶-AA⁷   I-2wherein AA¹-AA⁷ are as defined in Formula I.

In some examples of Formula I, m and n are 1, p is 0, and the compoundsare of Formula I-3:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶-AA⁷-AA⁸   I-3wherein AA¹-AA⁸ are as defined in Formula I.

In some examples of Formula I, m, n, and p are 1, and the compounds areof Formula I-4:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶-AA⁷-AA⁸-AA⁹   I-4wherein AA¹-AA⁹ are as defined in Formula I.

In some examples, the cell penetrating peptide moiety is cyclic, and thecompounds can be of Formula Ia:

wherein AA¹-AA⁹, m, n, and p are as defined in Formula I, and whereinthe curved line indicates a covalent bond.

In some examples of Formula Ia, m, n, and p are 0 and the compounds areof Formula Ia-1:

wherein AA¹-AA⁶ are as defined in Formula I.

In some examples of Formula Ia, m is 1, and n and p are 0, and thecompounds are of Formula Ia-2:

wherein AA¹-AA⁷ are as defined in Formula I.

In some examples of Formula Ia, m and n are 1, p is 0, and the compoundsare of Formula Ia-3:

wherein AA¹-AA⁸ are as defined in Formula I.

In some examples of Formula Ia, m, n, and p are 1, and the compounds areof Formula Ia-4:

wherein AA¹-AA⁹ are as defined in Formula I.

In some examples, the compound further comprises a cargo moiety, and thecompounds can be of Formula II:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶

AA⁷

_(m)

AA⁸

_(n)

AA⁹

_(p)car_(g)o   IIwherein the cargo moiety can comprise a detectable moiety, a therapeuticmoiety, a targeting moiety, or a combination thereof and AA¹-AA⁹, m, n,and p are as defined in Formula I.

In some examples of Formula II, m, n, and p are 0 and the compounds areof Formula II-1:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶-car_(g)o   II-1wherein AA¹-AA⁶ are as defined in Formula I and cargo is as defined inFormula II.

In some examples of Formula II, m is 1, and n and p are 0, and thecompounds are of Formula II-2:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶-AA⁷-car_(g)o   II-2wherein AA¹-AA⁷ are as defined in Formula I and cargo is as defined inFormula II.

In some examples of Formula II, m and n are 1, p is 0, and the compoundsare of Formula II-3:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶-AA⁷-AA⁸-car_(g)o   II-3wherein AA¹-AA⁸ are as defined in Formula I and cargo is as defined inFormula II.

In some examples of Formula II, m, n, and p are 1, and the compounds areof Formula II-4:AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶-AA⁷-AA⁸-AA⁹-car_(g)o   II-4wherein AA¹-AA⁹ are as defined in Formula I and cargo is as defined inFormula II.

In some examples, the cell penetrating peptide moiety and cargo moietytogether are cyclic, and the compounds are of Formula IIa:

wherein the cargo moiety is as defined in Formula II and AA¹-AA⁹, m, nand p are as defined in Formula I.

In some examples of Formula IIa, m, n, and p are 0 and the compounds areof Formula IIa-1:

wherein AA¹-AA⁶ are as defined in Formula I and cargo is as defined inFormula II. Also disclosed herein is Formula IIa-1 wherein one ofAA1-AA6 is absent (i.e., 5 amino acids in the endocyclic structure.

In some examples of Formula IIa, m is 1, and n and p are 0, and thecompounds are of Formula IIa-2:

wherein AA¹-AA⁷ are as defined in Formula I and cargo is as defined inFormula II.

In some examples of Formula IIa, m and n are 1, p is 0, and thecompounds are of Formula IIa-3:

wherein AA¹-AA⁸ are as defined in Formula I and cargo is as defined inFormula II.

In some examples of Formula IIa, m, n, and p are 1, and the compoundsare of Formula IIa-4:

wherein AA¹-AA⁹ are as defined in Formula I and cargo is as defined inFormula II.

In some examples, the cell penetrating peptide moiety is cyclic and thecargo moiety is appended to the cyclic cell penetrating peptide moietystructure, and the compounds are of Formula IIb:

wherein the cargo moiety is as defined in Formula II and AA¹-AA⁹, m, nand p are as defined in Formula I.

In some examples of Formula IIb, m, n, and p are 0 and the compounds areof Formula IIb-1:

wherein AA¹-AA⁶ are as defined in Formula I and cargo is as defined inFormula II.

In some examples of Formula IIb, m is 1, and n and p are 0, and thecompounds are of Formula IIb-2:

wherein AA¹-AA⁷ are as defined in Formula I and cargo is as defined inFormula II.

In some examples of Formula IIb, m and n are 1, p is 0, and thecompounds are of Formula IIb-3:

wherein AA¹-AA⁸ are as defined in Formula I and cargo is as defined inFormula II.

In some examples of Formula IIb, m, n, and p are 1, and the compoundsare of Formula IIb-4:

wherein AA¹-AA⁹ are as defined in Formula I and cargo is as defined inFormula II.

In some examples, the cargo moiety is cyclic and the cell penetratingpeptide moiety is cyclic, and together they form a fused bicyclicsystem, and the compounds are of Formula IIc:

wherein the cargo moiety is as defined in Formula II and AA¹-AA⁹, m, nand p are as defined in Formula I.

In some examples of Formula IIc, m, n, and p are 0 and the compounds areof Formula IIc-1:

wherein AA¹-AA⁶ are as defined in Formula I and cargo is as defined inFormula II.

In some examples of Formula IIc, m is 1, and n and p are 0, and thecompounds are of Formula IIc-2:

wherein AA¹-AA⁷ are as defined in Formula I and cargo is as defined inFormula II.

In some examples of Formula IIc, m and n are 1, p is 0, and thecompounds are of Formula IIc-3:

wherein AA¹-AA⁸ are as defined in Formula I and cargo is as defined inFormula II.

In some examples of Formula IIc, m, n, and p are 1, and the compoundsare of Formula IIc-4:

wherein AA¹-AA⁹ are as defined in Formula I and cargo is as defined inFormula II.

In some examples, the compounds can comprise any of the compounds inTable 6. Further examples are shown in Table 18 below.

TABLE 6 Example compounds SEQ ID NO Abbreviation Sequence  86 cFΦR₄^(Rho) cyclo(FΦRRRRQ)-K(Rho)  87 cFΦR₄ ^(Dex) cyclo(FΦRRRRQ)-K(Dex)  88Tat^(Dex) K(Dex)-GRKKRRQRRRPPQY  89 cFΦR₄ ^(FITC) cyclo(FΦRRRRQ)-K(FITC) 90 cFΦR₄-R₅ cyclo(FΦRRRRQ)-RRRRR-K(Rho)  91 cFΦR₄-A₅cyclo(FΦRRRRQ)-AAAAA-K(Rho)  92 cFΦR₄-F₄ cyclo(FΦRRRRQ)-FFFF-K(Rho)  93cFΦR₄-PCP cyclo(FΦRRRRQ)-miniPEG-DE(pCAP)LI  94 R₉-PCPRRRRRRRRR-miniPEG-DE(pCAP)LI  95 Tat-PCP RKKRRQRRR-miniPEG-DE(pCAP)LI 96 Antp-PCP RQIKIWFQNRRMKWKK-miniPEG-DE(pCAP)LI  97bicyclo(FΦR₄-A₅)^(Rho) [Tm(AAAAA)K(RRRRΦF)J]-K(Rho)  98bicyclo(FΦR₄-A₇)^(Rho) [Tm(AAAAAAA)K(RRRRΦF)J]-K(Rho)  99bicyclo(FΦR₄-RARAR)^(Rho) [Tm(RARAR)K(RRRRΦF)J]-K(Rho) 100bicyclo(FΦR₄-DADAD)^(Rho) [Tm(DADAD)K(RRRRΦF)J]-K(Rho) 101monocyclo(FΦR₄-A₅)^(Rho) cyclo(AAAAARRRRΦF)-K(Rho) 102monocyclo(FΦR₄-A₇)^(Rho) cyclo(AAAAAAARRRRΦF)-K(Rho) 103

104 CH₃CH₂CH₂CO⁻FΦRRRRUK(FITC) 105 DΩUD-Amc 106

107

108 CH₃CH₂CH₂CO⁻RRRRΦFDΩUD⁻A^(mc) 109 RRRRRRRRRDΩUC-Amc 110

111 FITC⁻URRRRFW_(Q)UTRV 112

113 cFΦR₄-PTP1B 114 cFΦR₄-PCP 115 cyclo((D-Thr)-(D-Asn)-(D-Val)-cyclo(tnvΛaRRRRΦ’FQ) F₂Pmp-(D-Ala)-Arg-Arg-Arg- Arg-Nal-Phe-Gln) 116cyclo(Ser-(D-Val)-Pro-F₂Pmp- cyclo(SvPΛHRRRR Φ’FQ)His-Arg-Arg-Arg-Arg-Nal-Phe- Gln) 117 cyclo(Ile-Pro-Phg-F₂Pmp-Nle-cyclo(IPΨΛΩRRRRΦ’FQ) Arg-Arg-Arg-Arg-Nal-Phe-Gln) 118cyclo((D-Ala)-Sar-(D-pThr)-Pp- cyclo(aΞt’

Φ’YQ)-K Nal-Tyr-Gln)-Lys 119 bicyclo[Tm((D-Ala)-Sar-(D- bicyclo[Tm(aΞ’

Φ’RA)J(FΦ’RRRRJ)]-K pThr)-Pp-Nal-Arg-Ala)-Dap- (Phe-Nal-Arg-Arg-Arg-Arg-Dap)]-Lys 120 bicyclo[Tm((D-Ala)-Sar-(D- bicyclo[Tm(aΞt’

Φ’RAa)J(FNΦ’RRRRJ)]-K pThr)-Pp-Nal-Arg-Ala-(D-Ala))-Dap-(Phe-Nal-Arg-Arg-Arg-Arg- Dap)]-Lys 121 bicyclo[Tm((D-Ala)-Sar-(D-bicyclo[Tm(aΞt

Φ’RAa)J(FΦ’RRRRJ)]-K Thr)-Pp-Nal-Arg-Ala-(D-Ala))-Dap-(Phe-Nal-Arg-Arg-Arg-Arg- Dap)]-Lys 122 bicyclo[Tm((D-Ala)-Sar-(D-bicyclo[Tm(aΞtaΦ’RAa)J(FΦ’RRRRJ)]-K Thr)-(D-Ala)-Nal-Arg-Ala-(D-Ala))-Dap-(Phe-Nal-Arg-Arg- Arg-Arg-Dap)]-Lys 123 Peptide 1

124 Peptide 2 CH₃CH₂CH₂CO-FΦRRRRUK(FITC)-NH₂ 125 Peptide 3 Ac-DMUD-Amc126 Peptide 4

127 Peptide 5

128 Peptide 6 CH₃CH₂CH₂CO⁻RRRRΦFDΩUD⁻A^(mc) 129 Peptide 7A^(c−)RRRRRRRRRDΩUD⁻A^(mc) 130 Peptide 8

131 Peptide 9 FITC-URRRRFWQUTRV-OH 132 Peptide 11

178 Monocyclic Inhibitor 1 cyclo(D-Thr-D-Asn-D-Val-F₂Pmp-D-Ala-Arg-Arg-Arg-Arg-Nal-Phe-Gln) 179 Monocyclic Inhibitor 2cyclo(Ser-D-Val-Pro-F₂Pmp-His-Arg-Arg- Arg-Arg-Nal-Phe-Gln) 180Monocyclic Inhibitor 3 cyclo(Ile-Pro-Phg-F₂Pmp-Nle-Arg-Arg-Arg-Arg-Nal-Phe-Gln) 181 Pin1 inhibitor 5cyclo(D-Ala-Sar-D-pThr-Pip-Nal-Tyr- Gln)-Lys-NH₂ 182 Pin1 inhibitor 6bicyclo[Tm(D-Ala-Sar-D-pThr-Pip-Nal- Arg-Ala)-Dap-(Phe-Nal-Arg-Arg-Arg-Arg-Dap)]-Lys-NH₂ 183 Pin1 inhibitor 7bicyclo[Tm(D-Ala-Sar-D-pThr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-Arg- Arg-Arg-Dap)]-Lys-NH₂ 184 Pin1inhibitor 8 bicyclo[Tm(D-Ala-Sar-D-Thr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-Arg- Arg-Arg-Dap)]-Lys-NH₂ 185 Pin1inhibitor 9 bicyclo[Tm(D-Ala-Sar-D-Thr-D-Ala-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg- Arg-Arg-Arg-Dap)]-Lys-NH₂ *Fpa, Σ:L-4-fluorophenylalanine; Pip, Θ: L-homoproline; Nle, Ω: L-norleucine;Phg, Ψ L-phenylglycine; F₂Pmp, Λ:L-4-(phosphonodifluoromethyl)phenylalanine; Dap, J:L-2,3-diaminopropionic acid; Nal, Φ’: L-β- naphthylalanine; Pp,

: L-pipecolic acid; Sar, Ξ: sarcosine; Tm, trimesic acid; Φ,L-2-naphthylalanine; Rho, rhodamine B; Dex, dexamethasone; FITC,fluorescein isothiocyanate; miniPEG, 8-amine-3,6-dioxaoctanoic acid;pCAP, phosphocoumaryl amino propionic acid; Amc,7-amino-4-methylcourmarin; FITC, fluorescein isothiocyanate; U,2-aminobutyric acid.

TABLE 7 Previously reported cell penetrating peptides SEQ ID NOAbbreviation Sequence 133 R₉ RRRRRRRRR 134 Tat YGRKKRRQRRR 135 AntpRQIKIWFQNRRMKWKK

Also disclosed herein are compositions comprising the compoundsdescribed herein.

Also disclosed herein are pharmaceutically-acceptable salts and prodrugsof the disclosed compounds. Pharmaceutically-acceptable salts includesalts of the disclosed compounds that are prepared with acids or bases,depending on the particular substituents found on the compounds. Underconditions where the compounds disclosed herein are sufficiently basicor acidic to form stable nontoxic acid or base salts, administration ofthe compounds as salts can be appropriate. Examples ofpharmaceutically-acceptable base addition salts include sodium,potassium, calcium, ammonium, or magnesium salt. Examples ofphysiologically-acceptable acid addition salts include hydrochloric,hydrobromic, nitric, phosphoric, carbonic, sulfuric, and organic acidslike acetic, propionic, benzoic, succinic, fumaric, mandelic, oxalic,citric, tartaric, malonic, ascorbic, alpha-ketoglutaric,alpha-glycophosphoric, maleic, tosyl acid, methanesulfonic, and thelike. Thus, disclosed herein are the hydrochloride, nitrate, phosphate,carbonate, bicarbonate, sulfate, acetate, propionate, benzoate,succinate, fumarate, mandelate, oxalate, citrate, tartarate, malonate,ascorbate, alpha-ketoglutarate, alpha-glycophosphate, maleate, tosylate,and mesylate salts. Pharmaceutically acceptable salts of a compound canbe obtained using standard procedures well known in the art, forexample, by reacting a sufficiently basic compound such as an amine witha suitable acid affording a physiologically acceptable anion. Alkalimetal (for example, sodium, potassium or lithium) or alkaline earthmetal (for example calcium) salts of carboxylic acids can also be made.

Methods of Making

The compounds described herein can be prepared in a variety of waysknown to one skilled in the art of organic synthesis or variationsthereon as appreciated by those skilled in the art. The compoundsdescribed herein can be prepared from readily available startingmaterials. Optimum reaction conditions can vary with the particularreactants or solvents used, but such conditions can be determined by oneskilled in the art.

Variations on the compounds described herein include the addition,subtraction, or movement of the various constituents as described foreach compound. Similarly, when one or more chiral centers are present ina molecule, the chirality of the molecule can be changed. Additionally,compound synthesis can involve the protection and deprotection ofvarious chemical groups. The use of protection and deprotection, and theselection of appropriate protecting groups can be determined by oneskilled in the art. The chemistry of protecting groups can be found, forexample, in Wuts and Greene, Protective Groups in Organic Synthesis, 4thEd., Wiley & Sons, 2006, which is incorporated herein by reference inits entirety.

The starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.),Sigma (St. Louis, Mo.), Pfizer (New York, N.Y.), GlaxoSmithKline(Raleigh, N.C.), Merck (Whitehouse Station, N.J.), Johnson & Johnson(New Brunswick, N.J.), Aventis (Bridgewater, N.J.), AstraZeneca(Wilmington, Del.), Novartis (Basel, Switzerland), Wyeth (Madison,N.J.), Bristol-Myers-Squibb (New York, N.Y.), Roche (Basel,Switzerland), Lilly (Indianapolis, Ind.), Abbott (Abbott Park, Ill.),Schering Plough (Kenilworth, N.J.), or Boehringer Ingelheim (Ingelheim,Germany), or are prepared by methods known to those skilled in the artfollowing procedures set forth in references such as Fieser and Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989). Othermaterials, such as the pharmaceutical carriers disclosed herein can beobtained from commercial sources.

Reactions to produce the compounds described herein can be carried outin solvents, which can be selected by one of skill in the art of organicsynthesis. Solvents can be substantially nonreactive with the startingmaterials (reactants), the intermediates, or products under theconditions at which the reactions are carried out, i.e., temperature andpressure. Reactions can be carried out in one solvent or a mixture ofmore than one solvent. Product or intermediate formation can bemonitored according to any suitable method known in the art. Forexample, product formation can be monitored by spectroscopic means, suchas nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C) infraredspectroscopy, spectrophotometry (e.g., UV-visible), or massspectrometry, or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

The disclosed compounds can be prepared by solid phase peptide synthesiswherein the amino acid α-N-terminal is protected by an acid or baseprotecting group. Such protecting groups should have the properties ofbeing stable to the conditions of peptide linkage formation while beingreadily removable without destruction of the growing peptide chain orracemization of any of the chiral centers contained therein. Suitableprotecting groups are 9-fluorenylmethyloxycarbonyl (Fmoc),t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz),biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl,2-cyano-t-butyloxycarbonyl, and the like. The9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularlypreferred for the synthesis of the disclosed compounds. Other preferredside chain protecting groups are, for side chain amino groups likelysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc),nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, andadamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxy-carbonyl,2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyland acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; forhistidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl;for tryptophan, formyl; for asparticacid and glutamic acid, benzyl andt-butyl and for cysteine, triphenylmethyl (trityl). In the solid phasepeptide synthesis method, the α-C-terminal amino acid is attached to asuitable solid support or resin. Suitable solid supports useful for theabove synthesis are those materials which are inert to the reagents andreaction conditions of the stepwise condensation-deprotection reactions,as well as being insoluble in the media used. Solid supports forsynthesis of α-C-terminal carboxy peptides is4-hydroxymethylphenoxymethyl-copoly(styrene-1% divinylbenzene) or4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resinavailable from Applied Biosystems (Foster City, Calif.). Theα-C-terminal amino acid is coupled to the resin by means ofN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC)or O-benzotriazol-1-yl-N,N,N,N-tetramethyluroniumhexafluorophosphate(HBTU), with or without 4-dimethylaminopyridine (DMAP),1-hydroxybenzotriazole (HOBT),benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate(BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), mediatedcoupling for from about 1 to about 24 hours at a temperature of between10° C. and 50° C. in a solvent such as dichloromethane or DMF. When thesolid support is4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin,the Fmoc group is cleaved with a secondary amine, preferably piperidine,prior to coupling with the α-C-terminal amino acid as described above.One method for coupling to the deprotected 4(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin isO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. Thecoupling of successive protected amino acids can be carried out in anautomatic polypeptide synthesizer. In one example, the α-N-terminal inthe amino acids of the growing peptide chain are protected with Fmoc.The removal of the Fmoc protecting group from the α-N-terminal side ofthe growing peptide is accomplished by treatment with a secondary amine,preferably piperidine. Each protected amino acid is then introduced inabout 3-fold molar excess, and the coupling is preferably carried out inDMF. The coupling agent can beO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.). At the endof the solid phase synthesis, the polypeptide is removed from the resinand deprotected, either in successively or in a single operation.Removal of the polypeptide and deprotection can be accomplished in asingle operation by treating the resin-bound polypeptide with a cleavagereagent comprising thianisole, water, ethanedithiol and trifluoroaceticacid. In cases wherein the α-C-terminal of the polypeptide is analkylamide, the resin is cleaved by aminolysis with an alkylamine.Alternatively, the peptide can be removed by transesterification, e.g.with methanol, followed by aminolysis or by direct transamidation. Theprotected peptide can be purified at this point or taken to the nextstep directly. The removal of the side chain protecting groups can beaccomplished using the cleavage cocktail described above. The fullydeprotected peptide can be purified by a sequence of chromatographicsteps employing any or all of the following types: ion exchange on aweakly basic resin (acetate form); hydrophobic adsorption chromatographyon underivitized polystyrene-divinylbenzene (for example, AmberliteXAD); silica gel adsorption chromatography; ion exchange chromatographyon carboxymethylcellulose; partition chromatography, e.g. on SephadexG-25, LH-20 or countercurrent distribution; high performance liquidchromatography (HPLC), especially reverse-phase HPLC on octyl- oroctadecylsilyl-silica bonded phase column packing.

Methods of Use

Also provided herein are methods of use of the compounds or compositionsdescribed herein. Also provided herein are methods for treating adisease or pathology in a subject in need thereof comprisingadministering to the subject an effective amount of any of the compoundsor compositions described herein.

Also provided herein are methods of treating, preventing, orameliorating cancer in a subject. The methods include administering to asubject an effective amount of one or more of the compounds orcompositions described herein, or a pharmaceutically acceptable saltthereof. The compounds and compositions described herein orpharmaceutically acceptable salts thereof are useful for treating cancerin humans, e.g., pediatric and geriatric populations, and in animals,e.g., veterinary applications. The disclosed methods can optionallyinclude identifying a patient who is or can be in need of treatment of acancer. Examples of cancer types treatable by the compounds andcompositions described herein include bladder cancer, brain cancer,breast cancer, colorectal cancer, cervical cancer, gastrointestinalcancer, genitourinary cancer, head and neck cancer, lung cancer, ovariancancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer,and testicular cancer. Further examples include cancer and/or tumors ofthe anus, bile duct, bone, bone marrow, bowel (including colon andrectum), eye, gall bladder, kidney, mouth, larynx, esophagus, stomach,testis, cervix, mesothelioma, neuroendocrine, penis, skin, spinal cord,thyroid, vagina, vulva, uterus, liver, muscle, blood cells (includinglymphocytes and other immune system cells). Further examples of cancerstreatable by the compounds and compositions described herein includecarcinomas, Karposi's sarcoma, melanoma, mesothelioma, soft tissuesarcoma, pancreatic cancer, lung cancer, leukemia (acute lymphoblastic,acute myeloid, chronic lymphocytic, chronic myeloid, and other), andlymphoma (Hodgkin's and non-Hodgkin's), and multiple myeloma.

The methods of treatment or prevention of cancer described herein canfurther include treatment with one or more additional agents (e.g., ananti-cancer agent or ionizing radiation). The one or more additionalagents and the compounds and compositions or pharmaceutically acceptablesalts thereof as described herein can be administered in any order,including simultaneous administration, as well as temporally spacedorder of up to several days apart. The methods can also include morethan a single administration of the one or more additional agents and/orthe compounds and compositions or pharmaceutically acceptable saltsthereof as described herein. The administration of the one or moreadditional agents and the compounds and compositions or pharmaceuticallyacceptable salts thereof as described herein can be by the same ordifferent routes. When treating with one or more additional agents, thecompounds and compositions or pharmaceutically acceptable salts thereofas described herein can be combined into a pharmaceutical compositionthat includes the one or more additional agents.

For example, the compounds or compositions or pharmaceuticallyacceptable salts thereof as described herein can be combined into apharmaceutical composition with an additional anti-cancer agent, such as13-cis-Retinoic Acid, 2-Amino-6-Mercaptopurine, 2-CdA,2-Chlorodeoxyadenosine, 5-fluorouracil, 6-Thioguanine, 6-Mercaptopurine,Accutane, Actinomycin-D, Adriamycin, Adrucil, Agrylin, Ala-Cort,Aldesleukin, Alemtuzumab, Alitretinoin, Alkaban-AQ, Alkeran,All-transretinoic acid, Alpha interferon, Altretamine, Amethopterin,Amifostine, Aminoglutethimide, Anagrelide, Anandron, Anastrozole,Arabinosylcytosine, Aranesp, Aredia, Arimidex, Aromasin, Arsenictrioxide, Asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab,Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib,Busulfan, Busulfex, C225, Calcium Leucovorin, Campath, Camptosar,Camptothecin-11, Capecitabine, Carac, Carboplatin, Carmustine,Carmustine wafer, Casodex, CCNU, CDDP, CeeNU, Cerubidine, cetuximab,Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone,Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabineliposomal, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin, Darbepoetinalfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride, Daunorubicinliposomal, DaunoXome, Decadron, Delta-Cortef, Deltasone, Denileukindiftitox, DepoCyt, Dexamethasone, Dexamethasone acetate, Dexamethasonesodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel,Doxil, Doxorubicin, Doxorubicin liposomal, Droxia, DTIC, DTIC-Dome,Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt, Epirubicin,Epoetin alfa, Erbitux, Erwinia L-asparaginase, Estramustine, Ethyol,Etopophos, Etoposide, Etoposide phosphate, Eulexin, Evista, Exemestane,Fareston, Faslodex, Femara, Filgrastim, Floxuridine, Fludara,Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream),Fluoxymesterone, Flutamide, Folinic Acid, FUDR, Fulvestrant, G-CSF,Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec, Lupron,Lupron Depot, Matulane, Maxidex, Mechlorethamine, -MechlorethamineHydrochlorine, Medralone, Medrol, Megace, Megestrol, Megestrol Acetate,Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, MethotrexateSodium, Methylprednisolone, Mylocel, Letrozole, Neosar, Neulasta,Neumega, Neupogen, Nilandron, Nilutamide, Nitrogen Mustard, Novaldex,Novantrone, Octreotide, Octreotide acetate, Oncospar, Oncovin, Ontak,Onxal, Oprevelkin, Orapred, Orasone, Oxaliplatin, Paclitaxel,Pamidronate, Panretin, Paraplatin, Pediapred, PEG Interferon,Pegaspargase, Pegfilgrastim, PEG-INTRON, PEG-L-asparaginase,Phenylalanine Mustard, Platinol, Platinol-AQ, Prednisolone, Prednisone,Prelone, Procarbazine, PROCRIT, Proleukin, Prolifeprospan 20 withCarmustine implant, Purinethol, Raloxifene, Rheumatrex, Rituxan,Rituximab, Roveron-A (interferon alfa-2a), Rubex, Rubidomycinhydrochloride, Sandostatin, Sandostatin LAR, Sargramostim, Solu-Cortef,Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin, Taxol,Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide,Thalomid, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide,Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab,Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid,Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs,Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP-16, Vumon,Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid, Zometa,Gliadel wafer, Glivec, GM-CSF, Goserelin, granulocyte colony stimulatingfactor, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine,HMM, Hycamtin, Hydrea, Hydrocort Acetate, Hydrocortisone, Hydrocortisonesodium phosphate, Hydrocortisone sodium succinate, Hydrocortonephosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin,Idarubicin, Ifex, IFN-alpha, Ifosfamide, IL 2, IL-11, Imatinib mesylate,Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEGconjugate), Interleukin 2, Interleukin-11, Intron A (interferonalfa-2b), Leucovorin, Leukeran, Leukine, Leuprolide, Leurocristine,Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine, L-PAM, L-Sarcolysin,Meticorten, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol, MTC, MTX,Mustargen, Mustine, Mutamycin, Myleran, Iressa, Irinotecan,Isotretinoin, Kidrolase, Lanacort, L-asparaginase, and LCR. Theadditional anti-cancer agent can also include biopharmaceuticals suchas, for example, antibodies.

Many tumors and cancers have viral genome present in the tumor or cancercells. For example, Epstein-Barr Virus (EBV) is associated with a numberof mammalian malignancies. The compounds disclosed herein can also beused alone or in combination with anticancer or antiviral agents, suchas ganciclovir, azidothymidine (AZT), lamivudine (3TC), etc., to treatpatients infected with a virus that can cause cellular transformationand/or to treat patients having a tumor or cancer that is associatedwith the presence of viral genome in the cells. The compounds disclosedherein can also be used in combination with viral based treatments ofoncologic disease.

Also described herein are methods of killing a tumor cell in a subject.The method includes contacting the tumor cell with an effective amountof a compound or composition as described herein, and optionallyincludes the step of irradiating the tumor cell with an effective amountof ionizing radiation. Additionally, methods of radiotherapy of tumorsare provided herein. The methods include contacting the tumor cell withan effective amount of a compound or composition as described herein,and irradiating the tumor with an effective amount of ionizingradiation. As used herein, the term ionizing radiation refers toradiation comprising particles or photons that have sufficient energy orcan produce sufficient energy via nuclear interactions to produceionization. An example of ionizing radiation is x-radiation. Aneffective amount of ionizing radiation refers to a dose of ionizingradiation that produces an increase in cell damage or death whenadministered in combination with the compounds described herein. Theionizing radiation can be delivered according to methods as known in theart, including administering radiolabeled antibodies and radioisotopes.

The methods and compounds as described herein are useful for bothprophylactic and therapeutic treatment. As used herein the term treatingor treatment includes prevention; delay in onset; diminution,eradication, or delay in exacerbation of signs or symptoms after onset;and prevention of relapse. For prophylactic use, a therapeuticallyeffective amount of the compounds and compositions or pharmaceuticallyacceptable salts thereof as described herein are administered to asubject prior to onset (e.g., before obvious signs of cancer), duringearly onset (e.g., upon initial signs and symptoms of cancer), or afteran established development of cancer. Prophylactic administration canoccur for several days to years prior to the manifestation of symptomsof an infection. Prophylactic administration can be used, for example,in the chemopreventative treatment of subjects presenting precancerouslesions, those diagnosed with early stage malignancies, and forsubgroups with susceptibilities (e.g., family, racial, and/oroccupational) to particular cancers. Therapeutic treatment involvesadministering to a subject a therapeutically effective amount of thecompounds and compositions or pharmaceutically acceptable salts thereofas described herein after cancer is diagnosed.

In some examples of the methods of treating of treating, preventing, orameliorating cancer or a tumor in a subject, the compound or compositionadministered to the subject can comprise a therapeutic moiety that cancomprise a targeting moiety that can act as an inhibitor against Ras(e.g., K-Ras), PTP1B, Pin1, Grb2 SH2, or combinations thereof.

The disclosed subject matter also concerns methods for treating asubject having a metabolic disorder or condition. In one embodiment, aneffective amount of one or more compounds or compositions disclosedherein is administered to a subject having a metabolic disorder and whois in need of treatment thereof. In some examples, the metabolicdisorder can comprise type II diabetes. In some examples of the methodsof treating of treating, preventing, or ameliorating the metabolicdisorder in a subject, the compound or composition administered to thesubject can comprise a therapeutic moiety that can comprise a targetingmoiety that can act as an inhibitor against PTP1B. In one particularexample of this method the subject is obese and the method comprisestreating the subject for obesity by administering a composition asdisclosed herein.

The disclosed subject matter also concerns methods for treating asubject having an immune disorder or condition. In one embodiment, aneffective amount of one or more compounds or compositions disclosedherein is administered to a subject having an immune disorder and who isin need of treatment thereof. In some examples of the methods oftreating of treating, preventing, or ameliorating the immune disorder ina subject, the compound or composition administered to the subject cancomprise a therapeutic moiety that can comprise a targeting moiety thatcan act as an inhibitor against Pin1.

The disclosed subject matter also concerns methods for treating asubject having cystic fibrosis. In one embodiment, an effective amountof one or more compounds or compositions disclosed herein isadministered to a subject having cystic fibrosis and who is in need oftreatment thereof. In some examples of the methods of treating thecystic fibrosis in a subject, the compound or composition administeredto the subject can comprise a therapeutic moiety that can comprise atargeting moiety that can act as an inhibitor against CAL PDZ.

Compositions, Formulations and Methods of Administration

In vivo application of the disclosed compounds, and compositionscontaining them, can be accomplished by any suitable method andtechnique presently or prospectively known to those skilled in the art.For example, the disclosed compounds can be formulated in aphysiologically- or pharmaceutically-acceptable form and administered byany suitable route known in the art including, for example, oral, nasal,rectal, topical, and parenteral routes of administration. As usedherein, the term parenteral includes subcutaneous, intradermal,intravenous, intramuscular, intraperitoneal, and intrasternaladministration, such as by injection. Administration of the disclosedcompounds or compositions can be a single administration, or atcontinuous or distinct intervals as can be readily determined by aperson skilled in the art.

The compounds disclosed herein, and compositions comprising them, canalso be administered utilizing liposome technology, slow releasecapsules, implantable pumps, and biodegradable containers. Thesedelivery methods can, advantageously, provide a uniform dosage over anextended period of time. The compounds can also be administered in theirsalt derivative forms or crystalline forms.

The compounds disclosed herein can be formulated according to knownmethods for preparing pharmaceutically acceptable compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E.W. Martin (1995)describes formulations that can be used in connection with the disclosedmethods. In general, the compounds disclosed herein can be formulatedsuch that an effective amount of the compound is combined with asuitable carrier in order to facilitate effective administration of thecompound. The compositions used can also be in a variety of forms. Theseinclude, for example, solid, semi-solid, and liquid dosage forms, suchas tablets, pills, powders, liquid solutions or suspension,suppositories, injectable and infusible solutions, and sprays. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions also preferably includeconventional pharmaceutically-acceptable carriers and diluents which areknown to those skilled in the art. Examples of carriers or diluents foruse with the compounds include ethanol, dimethyl sulfoxide, glycerol,alumina, starch, saline, and equivalent carriers and diluents. Toprovide for the administration of such dosages for the desiredtherapeutic treatment, compositions disclosed herein can advantageouslycomprise between about 0.1% and 100% by weight of the total of one ormore of the subject compounds based on the weight of the totalcomposition including carrier or diluent.

Formulations suitable for administration include, for example, aqueoussterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient; and aqueous and nonaqueous sterilesuspensions, which can include suspending agents and thickening agents.The formulations can be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and can be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions can be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, thecompositions disclosed herein can include other agents conventional inthe art having regard to the type of formulation in question.

Compounds disclosed herein, and compositions comprising them, can bedelivered to a cell either through direct contact with the cell or via acarrier means. Carrier means for delivering compounds and compositionsto cells are known in the art and include, for example, encapsulatingthe composition in a liposome moiety. Another means for delivery ofcompounds and compositions disclosed herein to a cell comprisesattaching the compounds to a protein or nucleic acid that is targetedfor delivery to the target cell. U.S. Pat. No. 6,960,648 and U.S.Application Publication Nos. 20030032594 and 20020120100 disclose aminoacid sequences that can be coupled to another composition and thatallows the composition to be translocated across biological membranes.U.S. Application Publication No. 20020035243 also describes compositionsfor transporting biological moieties across cell membranes forintracellular delivery. Compounds can also be incorporated intopolymers, examples of which include poly (D-L lactide-co-glycolide)polymer for intracranial tumors; poly[bis(p-carboxyphenoxy)propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL);chondroitin; chitin; and chitosan.

For the treatment of oncological disorders, the compounds disclosedherein can be administered to a patient in need of treatment incombination with other antitumor or anticancer substances and/or withradiation and/or photodynamic therapy and/or with surgical treatment toremove a tumor. These other substances or treatments can be given at thesame as or at different times from the compounds disclosed herein. Forexample, the compounds disclosed herein can be used in combination withmitotic inhibitors such as taxol or vinblastine, alkylating agents suchas cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracilor hydroxyurea, DNA intercalators such as adriamycin or bleomycin,topoisomerase inhibitors such as etoposide or camptothecin,antiangiogenic agents such as angiostatin, antiestrogens such astamoxifen, and/or other anti-cancer drugs or antibodies, such as, forexample, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN(Genentech, Inc.), respectively, or an immunotherapeutic such asipilimumab and bortezomib.

In certain examples, compounds and compositions disclosed herein can belocally administered at one or more anatomical sites, such as sites ofunwanted cell growth (such as a tumor site or benign skin growth, e.g.,injected or topically applied to the tumor or skin growth), optionallyin combination with a pharmaceutically acceptable carrier such as aninert diluent. Compounds and compositions disclosed herein can besystemically administered, such as intravenously or orally, optionallyin combination with a pharmaceutically acceptable carrier such as aninert diluent, or an assimilable edible carrier for oral delivery. Theycan be enclosed in hard or soft shell gelatin capsules, can becompressed into tablets, or can be incorporated directly with the foodof the patient's diet. For oral therapeutic administration, the activecompound can be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, aerosol sprays, and the like.

The disclosed compositions are bioavailable and can be delivered orally.Oral compositions can be tablets, troches, pills, capsules, and thelike, and can also contain the following: binders such as gumtragacanth, acacia, corn starch or gelatin; excipients such as dicalciumphosphate; a disintegrating agent such as corn starch, potato starch,alginic acid and the like; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, fructose, lactose or aspartame or aflavoring agent such as peppermint, oil of wintergreen, or cherryflavoring can be added. When the unit dosage form is a capsule, it cancontain, in addition to materials of the above type, a liquid carrier,such as a vegetable oil or a polyethylene glycol. Various othermaterials can be present as coatings or to otherwise modify the physicalform of the solid unit dosage form. For instance, tablets, pills, orcapsules can be coated with gelatin, wax, shellac, or sugar and thelike. A syrup or elixir can contain the active compound, sucrose orfructose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any unit dosage form should bepharmaceutically acceptable and substantially non-toxic in the amountsemployed. In addition, the active compound can be incorporated intosustained-release preparations and devices.

Compounds and compositions disclosed herein, including pharmaceuticallyacceptable salts or prodrugs thereof, can be administered intravenously,intramuscularly, or intraperitoneally by infusion or injection.Solutions of the active agent or its salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient, which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. The ultimatedosage form should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. Optionally, the prevention of the action of microorganismscan be brought about by various other antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the inclusion of agents that delay absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating a compoundand/or agent disclosed herein in the required amount in the appropriatesolvent with various other ingredients enumerated above, as required,followed by filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, compounds and agents disclosed herein can beapplied in as a liquid or solid. However, it will generally be desirableto administer them topically to the skin as compositions, in combinationwith a dermatologically acceptable carrier, which can be a solid or aliquid. Compounds and agents and compositions disclosed herein can beapplied topically to a subject's skin to reduce the size (and caninclude complete removal) of malignant or benign growths, or to treat aninfection site. Compounds and agents disclosed herein can be applieddirectly to the growth or infection site. Preferably, the compounds andagents are applied to the growth or infection site in a formulation suchas an ointment, cream, lotion, solution, tincture, or the like.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers, for example.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Useful dosages of the compounds and agents and pharmaceuticalcompositions disclosed herein can be determined by comparing their invitro activity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art.

The dosage ranges for the administration of the compositions are thoselarge enough to produce the desired effect in which the symptoms ordisorder are affected. The dosage should not be so large as to causeadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age,condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any counterindications. Dosage canvary, and can be administered in one or more dose administrations daily,for one or several days.

Also disclosed are pharmaceutical compositions that comprise a compounddisclosed herein in combination with a pharmaceutically acceptablecarrier. Pharmaceutical compositions adapted for oral, topical orparenteral administration, comprising an amount of a compound constitutea preferred aspect. The dose administered to a patient, particularly ahuman, should be sufficient to achieve a therapeutic response in thepatient over a reasonable time frame, without lethal toxicity, andpreferably causing no more than an acceptable level of side effects ormorbidity. One skilled in the art will recognize that dosage will dependupon a variety of factors including the condition (health) of thesubject, the body weight of the subject, kind of concurrent treatment,if any, frequency of treatment, therapeutic ratio, as well as theseverity and stage of the pathological condition.

Also disclosed are kits that comprise a compound disclosed herein in oneor more containers. The disclosed kits can optionally includepharmaceutically acceptable carriers and/or diluents. In one embodiment,a kit includes one or more other components, adjuncts, or adjuvants asdescribed herein. In another embodiment, a kit includes one or moreanti-cancer agents, such as those agents described herein. In oneembodiment, a kit includes instructions or packaging materials thatdescribe how to administer a compound or composition of the kit.Containers of the kit can be of any suitable material, e.g., glass,plastic, metal, etc., and of any suitable size, shape, or configuration.In one embodiment, a compound and/or agent disclosed herein is providedin the kit as a solid, such as a tablet, pill, or powder form. Inanother embodiment, a compound and/or agent disclosed herein is providedin the kit as a liquid or solution. In one embodiment, the kit comprisesan ampoule or syringe containing a compound and/or agent disclosedherein in liquid or solution form.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLES

The following examples are set forth to illustrate the methods andresults according to the disclosed subject matter. These examples arenot intended to be inclusive of all aspects of the subject matterdisclosed herein, but rather to illustrate representative methods andresults. These examples are not intended to exclude equivalents andvariations which are apparent to one skilled in the art.

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.) but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric. There are numerous variations and combinations ofreaction conditions, e.g., component concentrations, temperatures,pressures and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

Example 1

Cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR₄, where Φ isL-2-naphthylalanine) was found to be efficiently internalized bymammalian cells. In this study, its mechanism of internalization wasinvestigated by perturbing various endocytic events through theintroduction of pharmacologic agents and genetic mutations. The resultsshow that cFΦR₄ can bind directly to membrane phospholipids, can beinternalized into human cancer cells through endocytosis, and can escapefrom early endosomes into the cytoplasm. Its cargo capacity was examinedwith a wide variety of molecules including small-molecule dyes, linearand cyclic peptides of various charged states, and proteins. Dependingon the nature of the cargos, they may be delivered by endocyclic(insertion of cargo into the cFΦR₄ ring), exocyclic (attachment of cargoto the Gln side chain), or bicyclic approaches (fusion of cFΦR₄ andcyclic cargo rings). The overall delivery efficiency (i.e., delivery ofcargo into the cytoplasm and nucleus) of cFΦR₄ was 4-12-fold higher thanthose of nonaarginine (R₉), HIV Tat derived peptide (Tat), or penetratin(Antp). The higher delivery efficiency, coupled with superior serumstability, minimal toxicity, and synthetic accessibility, renders cFΦR₄a useful transporter for intracellular cargo delivery and a suitablesystem for investigating the mechanism of endosomal escape.

Introduction

The plasma membrane presents a major challenge in drug discovery,especially for biologics such as peptides, proteins and nucleic acids.One potential strategy to subvert the membrane barrier and deliver thebiologics into cells is to attach them to “cell-penetrating peptides(CPPs)”. Since the initial observation that HIV trans-activator oftranscription, Tat, internalizes into mammalian cells and activatesviral replication in the late 1980s (Frankel, A D and Pabo, C O. Cell,1988, 55, 1189-1193; Green, M and Loewenstein, P M. Cell, 1988, 55,1179-1188) a large number of CPPs consisting of 6-20 residues have beenreported (Langel, Ü. Cell-penetrating peptides: methods and protocols,Humana Press, New York, 2011, p xv; Schmidt, N et al. FEBS Lett., 2010,584, 1806-1813; Futaki, S. Adv. Drug Delivery Rev., 2005, 57, 547-558;Stewart, K M et al. Org. Biomol. Chem., 2008, 6, 2242-2255; Deshayes, Set al. Cell. Mol. Life Sci., 2005, 62, 1839-1849; Goun, E A et al.ChemBioChem, 2005, 7, 1497-1515). CPPs have been used to deliversmall-molecule drugs (Rothbard, J B et al. Nat. Med., 2000, 6,1253-1257; Nori, A et al. Bioconjugate Chem., 2003, 14, 44-50), DNA(Hoyer, J and Neundorf, I. Acc. Chem. Res., 2012, 45, 1048-1056; Eguchi,A et al. J. Biol. Chem., 2001, 276, 26204-26210), RNA (Nakase, I et al.Acc. Chem. Res., 2012, 45, 1132-1139; Andaloussi, S E et al. NucleicAcids Res., 2011, 39, 3972-3987; Jeong, J H et al. Bioconjugate Chem.,2009, 20, 5-14; Muratovska, A and Eccles, M R. FEBSLett., 2004, 558,63-68), proteins (Wadia, J S and Dowdy, S F. Adv. Drug Delivery Rev.,2005, 57, 579-596; Pooga, M et al. FASEB J., 2001, 15, 1451-1453;Schwarze, S R et al. Science, 1999, 285, 1569-1572), and nanoparticles(Josephson, L et al. Bioconjugate Chem., 1999, 10, 186-191; Gupta, B etal. Adv. Drug Delivery Rev., 2005, 57, 637-651; Liu, J et al.Biomacromolecules, 2001, 2, 362-8), into mammalian cells and tissuesthrough either covalent attachment or electrostatic association. ManyCPPs display minimal toxicity and immunogenicity at physiologicallyrelevant concentrations (Saar, K et al. Anal. Biochem., 2005, 345,55-65; Suhorutsenko, J et al. Bioconjugate Chem., 2011, 22, 2255-2262)and the incorporation of specific unnatural amino acids (Rueping, M etal. ChemBioChem, 2002, 3, 257-259) and other chemical moieties (Cooley,C B et al. J. Am. Chem. Soc., 2009, 131, 16401-16403; Pham, W et al.Chembiochem, 2004, 5, 1148-1151) have been found to increase stabilityand cytosolic delivery.

Despite three decades of investigation, the fundamental basis for CPPactivity remains elusive. Two distinct and non-mutually exclusivemechanisms have been proposed for the CPPs whose primary sequences arecharacterized by having multiple arginine residues. In the firstmechanism (direct membrane translocation), the arginine guanidiniumgroups interact with phospholipids of the plasma membrane to generateneutral ion pairs that passively diffuse across the membrane (Herce, H Dand Garcia, A E. Proc. Natl. Acad. Sci. U.S.A., 2007, 104, 20805-20810;Hirose, H et al. Mol. Ther., 2012, 20, 984-993) or promote the formationof transient pores that permit the CPPs to traverse the lipid bilayer(Herce, H D et al. Biophys. J., 2009, 97, 1917-1925; Palm-Apergi, C etal. FASEB J., 2009, 23, 214-223). In the second mechanism, CPPsassociate with cell surface glycoproteins and membrane phospholipids,internalize into cells through endocytosis (Richard, J P et al. J. Biol.Chem., 2005, 280, 15300-15306; Ferrari, A et al. Mol. Ther., 2003, 8,284-294; Fittipaldi, A et al. J. Biol. Chem., 2003, 278, 34141-34149;Kaplan, I M et al. J. Controlled Release, 2005, 102, 247-253; Nakase, Iet al. Biochemistry, 2007, 46, 492-501) and subsequently exit fromendosomes into the cytoplasm. Taken together, the majority of data showthat at low CPP concentrations, cellular uptake occurs mostly throughendocytosis, whereas direct membrane translocation becomes prevalent atconcentrations above 10 μM (Duchardt, F et al. Traffic, 2007, 8,848-866). However, the mechanism(s) of entry and the efficiency ofuptake may vary with the CPP identity, cargo, cell type, and otherfactors (Mueller, J et al. Bioconjugate Chem., 2008, 19, 2363-2374;Maiolo, J R et al. Biochim. Biophys. Acta., 2005, 1712, 161-172).

CPPs that enter cells via endocytosis must exit from endocytic vesiclesin order to reach the cytosol. Unfortunately, the endosomal membrane hasproven to be a significant barrier towards cytoplasmic delivery by theseCPPs; often a negligible fraction of the peptides escapes into the cellinterior (El-Sayed, A et al. AAPS J., 2009, 11, 13-22; Varkouhi, A K etal. J. Controlled Release, 2011, 151, 220-228; Appelbaum, J S et al.Chem. Biol., 2012, 19, 819-830). For example, even in the presence ofthe fusogenic hemagglutinin peptide HA2, which has been demonstrated toenhance endosomal cargo release, >99% of a Tat-Cre fusion proteinremains entrapped in macropinosomes 24 h after initial uptake (Kaplan, IM et al. J. Controlled Release, 2005, 102, 247-253). Recently, two newtypes of CPPs with improved endosomal escape efficiencies have beendiscovered. Appelbaum et al. showed that folded miniature proteinscontaining a discrete penta-arginine motif were able to effectivelyovercome endosomal entrapment and reach the cytosol of mammalian cells(Appelbaum, J S et al. Chem. Biol., 2012, 19, 819-830). This motifconsists of five arginines across three turns of an α-helix, andproteins containing this motif were released from early (Rab5⁺)endosomes into the cell interior. It has also been found thatcyclization of certain arginine-rich CPPs enhances their cellular uptake(Qian, Z et al. ACS Chem. Biol., 2013, 8, 423-431; Lattig-Tunnemann, Get al. Nat. Commun., 2011, 2, 453; Mandal, D et al. Angew. Chem. Int.Ed., 2011, 50, 9633-9637; Zhao, K et al. Soft Matter, 2012, 8,6430-6433). Small amphipathic cyclic peptides such as cyclo(FΦRRRRQ)(cFΦR₄, where Φ is L-2-naphthylalanine) are internalized by mammaliancells in an energy-dependent manner, and enter the cytoplasm and nucleuswith efficiencies 2-5-fold higher than that of nonaarginine (R₉) (Qian,Z et al. ACS Chem. Biol., 2013, 8, 423-431). Moreover, membraneimpermeable cargos such as phosphopeptides can be inserted into thecFΦR₄ ring resulting in their delivery into the cytoplasm of targetcells. However, insertion of a cargo into the cyclic peptide ring, whichis referred to herein as the “endocyclic” delivery method (FIG. 1A), islimited to relatively short peptides (≤7 amino acids), as large ringsdisplay poor internalization efficiency (Qian, Z et al. ACS Chem. Biol.,2013, 8, 423-431).

To gain insight into the cFΦR₄ mechanism of action and potentiallydesign cyclic CPPs of still higher efficiency, herein theinternalization mechanism of cFΦR₄ was investigated through the use ofartificial membranes and pharmacologic agents as well as geneticmutations that perturb various endocytic events. The data show thatcFΦR₄ can bind directly to the plasma membrane phospholipids and canenter cells through endocytosis. Like the miniature proteins displayingthe penta-arginine motif (Appelbaum, J S et al. Chem. Biol., 2012, 19,819-830), cFΦR₄ can escape from the early endosomes into the cytosol.The ability of cFΦR₄ to deliver a wide range of cargo molecules,including linear peptides of varying charges, cyclic peptides, and largeproteins, into the cytoplasm of mammalian cells by exocyclic (attachmentof cargo to the Gln side chain; FIG. 1B) or bicyclic delivery methods(fusion of the cFΦR₄ and cyclic cargo rings; FIG. 1C) was also examined.It was found that cFΦR₄ is tolerant to the size and nature of cargos andefficiently transported all of the cargos tested into the cytoplasm andnucleus of mammalian cells. In addition, cFΦR₄ exhibits superiorstability against proteolysis over linear CPPs but minimal cytotoxicity.cFΦR₄ therefore provides a practically useful transporter for cytosoliccargo delivery as well as a system for investigating the mechanism ofearly endosomal cargo release.

Materials.

Reagents for peptide synthesis were purchased from Advanced ChemTech(Louisville, Ky.), NovaBiochem (La Jolla, Calif.), or Anaspec (San Jose,Calif.). 2,2′-Dipyridyl disulfide, Lissamine rhodamine B sulfonylchloride, fluorescein isothiocyanate (FITC), dexamethasone (Dex),coenzyme A trilithium salt, FITC-labeled dextran (dextran^(FITC)) andhuman serum were purchased from Sigma-Aldrich (St. Louis, Mo.). Cellculture media, fetal bovine serum (FBS), penicillin-streptomycin, 0.25%trypsin-EDTA, Hoescht 33342, Alexa488-labeled dextran(dextran^(Alexa488)), Dulbecco's phosphate-buffered saline (DPBS) (2.67mM potassium chloride, 1.47 mM potassium phosphate monobasic, 137 mMsodium chloride, 8.06 mM sodium phosphate dibasic), and Lipofectamine2000 were purchased from Invitrogen (Carlsbad, Calif.). PD-10 desaltingcolumns were purchased from GE-Healthcare (Piscataway, N.J.). Nuclearstaining dye DRAQ5™ was purchased from Thermo Scientific (Rockford,Ill.), while cell proliferation kit (MTT) was purchased from Roche(Indianapolis, Ind.). Anti-phosphotyrosine (pY) antibody (clone 4G10)was purchased from Millipore (Temecula, Calif.).

Rink resin LS (100-200 mesh, 0.2 mmol/g) was purchased from AdvancedChemTech. LC-SMCC(succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate])was purchased from Thermo Scientific (Rockford, Ill.), while1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1′-rac-glycerol) (sodium salt)(POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phophoethanolamine (POPE),sphingomyelin (Brain, Porcine), and cholesterol were purchased fromAvanti Polar Lipids (Alabaster, Ala.). Heparan sulfate (HO-03103, Lot #HO-10697) was obtained from Celcus Laboratories (Cincinnati, Ohio).

Peptide Synthesis and Labeling.

Peptides were synthesized on Rink Resin LS (0.2 mmol/g) using standardFmoc chemistry. The typical coupling reaction contained 5 equiv ofFmoc-amino acid, 5 equiv of2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) and 10 equiv of diisopropylethylamine (DIPEA)and was allowed to proceed with mixing for 75 min. After the addition ofthe last (N-terminal) residue, the allyl group on the C-terminal Gluresidue was removed by treatment with Pd(PPh₃)₄ and phenylsilane (0.1and 10 equiv, respectively) in anhydrous DCM (3×15 min). The N-terminalFmoc group was removed by treatment with 20% piperidine in DMF and thepeptide was cyclized by treatment withbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP)/HOBt/DIPEA (5, 5, and 10 equiv) in DMF for 3 h. The peptideswere deprotected and released from the resin by treatment with82.5:5:5:5:2.5 (v/v) TFA/thioanisole/water/phenol/ethanedithiol for 2 h.The peptides were triturated with cold ethyl ether (3×) and purified byreversed-phase HPLC on a C18 column. The authenticity of each peptidewas confirmed by MALDI-TOF mass spectrometry.

Peptide labeling with FITC was performed by dissolving the purifiedpeptide (˜1 mg) in 300 μL of 1:1:1 (vol/vol) DMSO/DMF/150 mM sodiumbicarbonate (pH 8.5) and mixing with 10 μL of FITC in DMSO (100 mg/mL).After 20 min at room temperature, the reaction mixture was subjected toreversed-phase HPLC on a C18 column to isolate the FITC-labeled peptide.To generate rhodamine- and Dex-labeled peptides (FIG. 2), anN^(ε)-4-methoxytrityl-L-lysine was added to the C-terminus. After thesolid phase peptide synthesis, the lysine side chain was selectivelydeprotected using 1% (v/v) trifluoroacetic acid in CH₂Cl₂. The resin wasincubated with Lissamine rhodamine B sulfonyl chloride/DIPEA (5 equiveach) in DMF overnight. The peptides were fully deprotected, trituratedwith diethyl ether, and purified by HPLC. The Dex-labeled peptide wasproduced by incubating the resin with dexamethasone-21-thiopropionicacid/HBTU/DIPEA (5, 5, and 10 equiv) in DMF for 3 h (Appelbaum, J S etal. Chem. Biol., 2012, 19, 819-830). The peptide was then deprotected,triturated, and purified by HPLC. Bicyclic peptides, phosphocoumarylaminopropionic acid (pCAP), and pCAP-containing peptides (PCPs) weresynthesized as previously described (Lian, W et al. J. Am. Chem. Soc.,2013, 135, 11990-11995; Mitra, S and Barrios, A M. Bioorg. Med. Chem.Lett., 2005, 15, 5124-5145; Stanford, S M et al. Proc. Natl. Acad. Sci.U.S.A., 2012, 109, 13972-13977). The authenticity of each peptide wasconfirmed by MALDI-TOF mass spectrometry.

Preparation of cFΦR₄-Protein Conjugates.

The gene coding for the catalytic domain of PTP1B (amino acids 1-321)was amplified by the polymerase chain reaction using PTP1B cDNA astemplate and oligonucleotides5′-ggaattccatatggagatggaaaaggagttcgagcag-3′ and5′-gggatccgtcgacattgtgtggctccaggattcgtftgg-3′ as primers. The resultingDNA fragment was digested with endonucleases Nde I and Sal I andinserted into prokaryotic vector pET-22b(+)-ybbR (Yin, J et al. Proc.Natl. Acad. Sci. U.S.A., 2005, 102, 15815-15820). This cloning procedureresulted in the addition of a ybbR tag (VLDSLEFIASKL) to the N-terminusof PTP1B. Expression and purification of the ybbR tagged PTP1B werecarried out as previously described (Ren, L et al. Biochemistry, 2011,50, 2339-2356).

Peptide cFΦR₄ containing a C-terminal cysteine (cFΦR₄-SH, ˜10 μmol; FIG.3) was dissolved in 1 mL of degassed DPBS and mixed with 2,2′-dipyridyldisulfide (5 equiv) dissolved in acetone (0.5 mL). After 2 h at roomtemperature, the reaction product cFΦR₄-SS-Py was purified byreversed-phase HPLC. The product was incubated with coenzyme A (2 equiv)in DPBS for 2 h. The resulting cFΦR₄-SS-CoA adduct was purified again byreversed-phase HPLC. Green fluorescent protein (GFP) containing anN-terminal ybbR tag (VLDSLEFIASKL) and a C-terminal six-histidine tagwas expressed in Escherichia coli and purified as previously described(Yin, J et al. Proc. Natl. Acad. Sci. U.S.A., 2005, 102, 15815-15820).Next, ybbR-GFP (30 μM), cFΦR₄-SS-CoA (30 μM), and phosphopantetheinyltransferase Sfp (0.5 μM) were mixed in 50 mM HEPES (pH 7.4), 10 mM MgCl2(total volume 1.5 mL) and incubated at 37° C. for 15 min. The labeledprotein, cFΦR₄-S-S-GFP (FIG. 3), was separated from unreactedcFΦR₄-SS-CoA by passing the reaction mixture through a PD-10 desaltingcolumn. GFP conjugated to Tat (Tat-S-S-GFP) and cFΦR₄-conjugated PTP1B(cFΦR₄-PTP1B) were prepared in a similar fashion (FIG. 4).

Peptide containing a C-terminal lysine (cFΦR₄-Lys, ˜10 μmol; FIG. 4) wassynthesized on the solid phase, deprotected and released from thesupport, dissolved in degassed DPBS (pH 7.4, 1 mL), and mixed withbifunctional linker LC-SMCC (5 equiv) dissolved in DMSO (0.2 mL). Afterincubation at room temperature for 2 h, the reaction product cFΦR₄-SMCC(FIG. 4) was purified by reversed-phase HPLC equipped with a C18 column.The product was then mixed with coenzyme A (2 equiv) in DPBS andincubated for 2 h. The resulting cFΦR₄-SMCC-CoA adduct was purifiedagain by reversed-phase HPLC. Next, ybbR-tagged PTP1B (30 μM),cFΦR₄-SMCC-CoA (30 μM), and phosphopantetheinyl transferase Sfp (0.5 μM)were mixed in 50 mM HEPES (pH 7.4), 10 mM MgCl₂ (total volume of 1.5 mL)and incubated at 37° C. for 15 min. The labeled protein (cFΦR₄-PTP1B;FIG. 4) was separated from unreacted cFΦR₄-SMCC-CoA by passing thereaction mixture through a PD-10 desalting column eluted with DPBS.

Cell Culture and Transfection.

HEK293, HeLa, MCF-7, NIH 3T3 and A549 cells were maintained in mediumconsisting of DMEM, 10% FBS and 1% penicillin/streptomycin. Jurkat,H1650, and H1299 cells were grown in RPMI-1640 supplemented with 10% FBSand 1% penicillin/streptomycin. Cells were cultured in a humidifiedincubator at 37° C. with 5% CO₂. For HeLa cells transfection, cells wereseeded onto 96-well plate at a density of 10,000 cells/well. Followingattachment, cells were transfected with plasmids encoding Rab5-greenfluorescent protein fusion (Rab5-GFP), Rab7-GFP (Addgene plasmid#28047), glucocorticoid receptor (C638G)-GFP fusion (GR-GFP) (Holub, J Met al. Biochemistry, 2013, 50, 9036-6046), DsRed-Rab5 WT (Addgeneplasmid #13050) or DsRed-Rab5^(Q79L) (Addgene plasmid #29688) followingLipofectamine 2000 manufacturer protocols.

Preparation of Small Unilamellar Vesicles (SUVs).

SUVs were prepared by modifying a previously reported procedure(Magzoub, M et al. Biochim. Biophys. Acta, 2002, 1563, 53-63). A properlipid mixture was dissolved in chloroform in a test tube. The lipidmixture was dried gently by blowing argon over the solution, and kept ina desiccator overnight. The dried lipids were rehydrated in DPBS tofinal total lipid concentration of 10 mM. The suspension was rigorouslymixed by vortexing and sonication on ice until it became clear. Atypical preparation yields a homogeneous solution containing vesicleswith average diameter of ˜80 nm and polydispersity (PdI) index of <0.15as determined by dynamic light scattering measurements using Zeta SizerNano Series (Malvern, Brookhaven, Conn.). The SUV solution was stored at4° C. and used for FP experiments on the same day.

Fluorescence Polarization.

A typical experiment was performed by incubating 100 nM FITC-labeledpeptide with varying concentrations of heparan sulfate (0-5,000 nM) inDPBS for 2 h at room temperature. The FP values were measured on aMolecular Devices Spectramax M5 spectrofluorimeter, with excitation andemission wavelengths at 485 and 525 nm, respectively. EC₅₀ weredetermined by plotting the FP values as a function of heparan sulfateconcentrations and fitted to a four-parameter logistic curve withGraphPad PRISM ver.6 software.

To obtain the EC50 value of CPP with lipid membranes, the FP experimentwas similarly conducted using 100 nM FITC-labeled peptide withincreasing concentrations of SUV solutions (0-10 mM) in DPBS. The FPvalues were similarly measured, plotted, and analyzed.

Image Analysis.

Raw images were uniformly modified using imageJ. Pearson's correlationcoefficient (R) was obtained from endosomal regions using Just AnotherColocalization Plugin (JACoP) (Bolte, S and Cordelieres, F P. J.Microsc., 2006, 224, 213-232). For GR-GFP translocation assay,individual GFP and Hoescht images were loaded into a customizedCellProfiler pipeline and colored to grey (Carpenter, A E et al. GenomeBiol., 2006, 7, R100). Nuclei were distinguished from the Hoescht imagevia Otsu automatic three-class thresholding, with pixels of the middleintensity class assigned to background. Clumped objects were identifiedusing Laplacian of Gaussian modeling and separated by shape. The nuclearregion was defined as the diameter of the Hoescht objects shrunken by 1μm, while the cytosolic ring region was defined as the region betweenthe nuclear diameter and the nuclear diameter expanded 2 μm. Thetranslocation ratio was defined as the mean GFP signal inside thenuclear region divided by the mean GFP signal within the cytosolicregion measured per cell, and 30-70 cells from 15-30 images werecaptured for each condition tested.

Confocal Microscopy.

To examine the co-localization between rhodamine-labeled cyclicpeptide)(cFΦR₄ ^(Rho)) and Rab5⁺ or Rab7⁺ endosomes, HeLa cellstransfected with Rab5-GFP or Rab7-GFP were plated (200 μL, 10⁴cells/well, 96-well glass bottom MatriPlates) the day prior to theexperiment. On the day of experiment, HeLa cells were treated with 1 μMcFΦR₄ ^(Rho) in DMEM media supplemented with 300 nM Hoescht 33342 for 30min. After that, the cells were washed with HKR buffer (10 mM HEPES, pH7.4, 140 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂) and imaged using aPerkinElmer LiveView spinning disk confocal microscope.

For GR translocation assay, HeLa cells transfected with GR-GFP wereplated as described above (Holub, J M et al. Biochemistry, 2013, 50,9036-6046). The cells were treated for 30 min with DMEM media containing1 μM Dex or Dex-peptide conjugate and 300 nM Hoescht 33342 and imagedusing a Zeiss Axiovert 200M epifluorescence microscope outfitted withZiess Axiocam mRM camera and an EXFO-Excite series 120 Hg arc lamp. Toexamine the effect of endocytosis inhibitors, transfected HeLa cellswere pretreated for 30 min with clear DMEM containing the inhibitorsbefore incubation with Dex or Dex-peptide conjugates. To test whetherRab5 activity is required for endosomal escape, HeLa cells weretransfected with GR-GFP and DsRed-Rab5 WT or DsRed-Rab5^(Q79L) beforetreatment with Dex or Dex-peptide conjugate and imaged as describedabove (Appelbaum, J S et al. Chem. Biol., 2012, 19, 819-830).

To examine the internalization of rhodamine-labeled peptides, 5×10⁴HEK293 cells were plated in a 35 mm glass-bottomed microwell dish(MatTek). On the day of experiment, the cells were incubated with thepeptide solution (5 μM) and 0.5 mg/mL dextran^(FITC) at 37° C. for 2 h.The cells were gently washed with DPBS twice and imaged on a VisitechInfinity 3 Hawk 2D-array live cell imaging confocal microscope. Todetect the internalization of pCAP-containing peptides, HEK293 cellswere similarly plated and incubated with the peptide solution (5 μM) at37° C. for 60 min. After removal of the medium, the cells were gentlywashed with DPBS containing sodium pervanadate (1 mM) twice andincubated for 10 min in DPBS containing 5 μM nuclear staining dye DRAQS.The resulting cells were washed with DPBS twice and imaged on a spinningdisk confocal microscope (UltraView Vox CSUX1 system). To monitor GFPinternalization, 5×10⁴ HEK293 cells were seeded in a 35 mmglass-bottomed microwell dish and cultured overnight. Cells were treatedwith cFΦR₄-S-S-GFP (1 μM) at 37° C. for 2 h. After removal of themedium, the cells were incubated in DPBS containing 5 μM DRAQS for 10min. The cells were washed with DPBS twice and imaged on a VisitechInfinity 3 Hawk 2D-array live cell imaging confocal microscope.

Flow Cytometry.

To quantify the delivery efficiencies of pCAP-containing peptides, HeLacells were cultured in six-well plates (5×10⁵ cells per well) for 24 h.On the day of experiment, the cells were incubated with 10 μMpCAP-containing peptide in clear DMEM with 1% FBS at 37° C. for 2 h. Thecells were washed with DPBS containing 1 mM sodium pervanadate, detachedfrom plate with 0.25% trypsin, suspended in DPBS containing 1% bovineserum albumin, and analyzed on a BD FACS Aria flow cytometer withexcitation at 355 nm. Data were analyzed with Flowjo software (TreeStar).

To estimate the effect of cFΦR₄ on endocytosis, HeLa cells were seededin six-well plates (5×10⁵ cells per well) and allowed to adhereovernight. Following adherence, cells were treated with clear DMEMcontaining no supplement, 1 μM cFΦR₄ peptide, 100 μM dextran^(Alexa488)(Life Technologies, D-22910), or both 1 μM cyclic peptide and 100 μMdextran^(Alexa488) for 30 min under standard cell culture conditions.The cells were washed with DPBS twice, removed from the plate with 0.25%trypsin, diluted into clear DMEM containing 10% FBS, pelleted at 300 gfor 5 min, washed once with DPBS and resuspended in 200 μL of DPBS.Whole-cell dextran uptake was analyzed on a BD Accuri C6 flow cytometerusing the manufacturer FL1 laser and filter set.

Immunoblotting.

NIH 3T3 cells were cultured in full growth media to reach 80%confluence. The cells were starved in serum free media for 3 h andtreated with different concentrations of cFΦR₄-PTP1B or untagged PTP1Bfor 2 h, followed by 30 min incubation in media supplemented with 1 mMsodium pervanadate. The solutions were removed and the cells were washedwith cold DPBS twice. The cells were detached and lysed in 50 mMTris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 10 mM sodium pyrophosphate, 5mM iodoacetic acid, 10 mM NaF, 1 mM EDTA, 2 mM sodium pervanadate, 0.1mg/mL phenylmethanesulfonyl fluoride, 1 mM benzamidine, and 0.1 mg/mLtrypsin inhibitor. After 30 min incubation on ice, the cell lysate wascentrifuged at 15,000 rpm for 25 min in a microcentrifuge. The totalcellular proteins were separated by SDS-PAGE and transferredelectrophoretically to PVDF membrane, which was immunoblotted usinganti-pY antibody 4G10.

Serum Stability Test.

The stability tests were carried by modifying a previously reportedprocedure (Nguyen, L T et al. PLoS One, 2010, 5, e12684). Diluted humanserum (25%) was centrifuged at 15,000 rpm for 10 min, and thesupernatant was collected. A peptide stock solution was diluted into thesupernatant to a final concentration of 5 μM for cFΦR₄ and Antp and 50μM for peptides R9 and Tat and incubated at 37° C. At various timepoints (0-6 h), 200-μL aliquots were withdrawn and mixed with 50 μl of15% trichloroacetic acid and incubated at 4° C. overnight. The finalmixture was centrifuged at 15,000 rpm for 10 min in a microcentrifuge,and the supernatant was analyzed by reversed-phase HPLC equipped with aC18 column (Waters). The amount of remaining peptide (%) was determinedby integrating the area underneath the peptide peak (monitored at 214nm) and compared with that of the control reaction (no serum).

Cytotoxicity Assay.

MTT assays were performed to evaluate cyclic peptide's cytotoxicityagainst several mammalian cell lines (Mosmann, T. J. Immunol. Methods,1983, 65, 55-63). One hundred μL of MCF-7, HEK293, H1299, H1650, A549(1×10⁵ cells/mL) cells were placed in each well of a 96-well cultureplate and allowed to grow overnight. Varying concentrations of thepeptide (5 or 50 μM) were added to the each well and the cells wereincubated at 37° C. with 5% CO₂ for 24 to 72 h. Ten μL of MTT stocksolution was added into each well. Addition of 10 μL of the solution tothe growth medium (no cell) was used as a negative control. The platewas incubated at 37° C. for 4 h. Then 100 μL of SDS-HCl solubilizingbuffer was added into each well, and the resulting solution was mixedthoroughly. The plate was incubated at 37° C. for another 4 h. Theabsorbance of the formazan product was measured at 570 nm using aMolecular Devices Spectramax M5 plate reader. Each experiment wasperformed in triplicates and the cells without any peptide added weretreated as control.

cFΦR₄ Binds to Membrane Phospholipids.

It was previously observed that incubation of 1 μM FITC-labeled cyclicpeptide cFΦR₄ ^(FITC) with vesicles containing negatively chargedphospholipids (90% phosphatidylcholine (PC) and 10% phosphatidylglycerol(PG)) resulted in quenching of the peptide fluorescence, consistent withdirect binding of cFΦR₄ to phospholipids (Qian, Z et al. ACS Chem.Biol., 2013, 8, 423-431). To test the potential role of membrane bindingduring endocytic uptake of CPPs, SUVs that mimic the outer membrane ofmammalian cells (45% PC, 20% phosphatidylethanolamine, 20%sphingomyelin, and 15% cholesterol) were prepared and tested for bindingto FITC-labeled cFΦR₄, R9, and Tat (each at 100 nM) by a fluorescencepolarization (FP) assay. cFΦR₄ bound to the neutral SUVs with an EC50value (lipid concentration at which half of cFΦR₄ ^(FITC) is bound) of2.1±0.1 mM (FIG. 5A). R9 showed much weaker binding to the artificialmembrane (EC50>10 mM), whereas Tat did not bind at all. Next, the CPPswere tested for binding to heparan sulfate, which was previouslyproposed to be the primary binding target of cationic CPPs (Nakase, I etal. Biochemistry, 2007, 46, 492-501; Rusnati, M et al. J. Biol. Chem.,1999, 274, 28198-28205; Tyagi, M et al. J. Biol. Chem., 2001, 276,3254-3261; Ziegler, A and Seelig, J. Biophys. J., 2004, 86, 254-263;Goncalves, E et al. Biochemistry, 2005, 44, 2692-2702; Ziegler, A. Adv.Drug Delivery Rev., 2008, 60, 580-597). R9 and Tat both bound to heparansulfate with high affinity, having EC50 values of 144 and 304 nM,respectively (FIG. 5B). Under the same condition, cFΦR₄ showed nodetectable binding to heparan sulfate. These results are in agreementwith the previous observations that non-amphipathic cationic CPPs (e.g.,Tat and R9) bind tightly with cell surface proteoglycans (e.g. heparansulfate) but only weakly with membrane lipids (Ziegler, A. Adv. DrugDelivery Rev., 2008, 60, 580-597). The insufficient number of positivecharges of cFΦR₄ is likely responsible for its lack of strongelectrostatic interaction with heparan sulfate. On the other hand, theamphipathic nature and the more rigid cyclic structure of cFΦR₄ shouldfacilitate its binding to neutral lipid membranes. These data, togetherwith the inhibition pattern by various endocytic inhibitors describedabove, suggest that cFΦR₄ can bind directly to the plasma membranephospholipids and can be internalized by all of the endocytic mechanismsin a piggyback manner.

Intracellular Delivery of Peptidyl Cargos.

Since endocyclic delivery by cFΦR₄ is limited to a heptapeptide orsmaller cargos (Qian, Z et al. ACS Chem. Biol., 2013, 8, 423-431), inthis study the ability of cFΦR₄ to deliver cargos of varying sizes andphysicochemical properties attached to the Gln side chain (FIG. 1B,exocyclic delivery) was tested. First, positively charged (RRRRR),neutral (AAAAA), hydrophobic (FFFF), and negatively charged peptides[DE(pCAP)LI] were covalently attached to cFΦR₄. The first three peptideswere labeled with rhodamine B at a C-terminal lysine side chain (FIG.2), and their internalization into HEK293 cells was examined bylive-cell confocal microscopy. Cells incubated for 2 h with 5 μM peptidecFΦR₄-A₅ (FIG. 6A) or cFΦR₄-R5 (FIG. 6B) showed evidence of bothpunctuate and diffuse fluorescence, with the latter distributed almostuniformly throughout the cell. In contrast, the fluid phase endocyticmarker dextran^(FITC) displayed predominantly punctuate fluorescence,indicative of endosomal localization. The diffuse rhodamine fluorescencesuggests that a fraction of the peptides reached the cytosol and nucleusof the cells. Co-incubation of cells with cFΦR₄ (1 μM) anddextran^(Alexa488) increased the internalization of the endocytic markerby 15% (FIG. 7), suggesting that cFΦR₄ can activate endocytosis incultured cells. cFΦR₄-F₄ was not tested due to its poor aqueoussolubility.

Peptide cFΦR4-DE(pCAP)LI (cFΦR4-PCP; FIG. 2) was designed to test theability of cFΦR₄ to deliver negatively charged cargos as well as tocompare the cytoplasmic delivery efficiency of cFΦR₄ with those of otherwidely used CPPs such as R9, Tat, and penetratin (Antp). Thus, untaggedPCP [Ac-DE(pCap)LI-NH₂] and PCP conjugated to R9 (R₉-PCP), Tat(Tat-PCP), or Antp (Antp-PCP) were also prepared. Note that cFΦR₄-PCPcarries a net charge of zero at physiological pH. pCAP isnon-fluorescent, but upon entering the cell interior, should be rapidlydephosphorylated by endogenous protein tyrosine phosphatases (PTPs) toproduce a fluorescent product, coumaryl aminopropionic acid (CAP,excitation 355 nm; emission 450 nm) (Mitra, S and Barrios, A M. Bioorg.Med. Chem. Lett., 2005, 15, 5124-5145; Stanford, S M et al. Proc. Natl.Acad. Sci. U.S.A., 2012, 109, 13972-13977). When assayed against a PTPpanel in vitro, all four CPP-PCP conjugates were efficientlydephosphorylated (Table 8). This assay detects only the CPP-cargo insidethe cytoplasm and nucleus, where the catalytic domains of all knownmammalian PTPs are localized (Alonso, A et al. Cell, 2004, 117,699-711). Further, CAP is fluorescent only in its deprotonated state(pKa=7.8); even if some dephosphorylation occurs inside the endosome (pH6.5-4.5) or lysosome (pH 4.5), it would contribute little to the totalfluorescence (FIG. 8). Treatment of HEK293 cells with 5 μM cFΦR₄-PCP for60 min resulted in diffuse blue fluorescence throughout the cell,suggesting that cFΦR₄-PCP reached the cell interior, whereas theuntagged PCP failed to enter cells under the same condition (FIG. 9A).When HEK293 cells were pretreated with the PTP inhibitor sodiumpervanadate for 1 h prior to incubation with cFΦR₄-PCP (5 μM), the CAPfluorescence in the cells diminished to background levels. HEK293 cellstreated with R₉-PCP, Antp-PCP, or Tat-PCP under identical conditionsshowed weak fluorescence, consistent with the poor ability of thesepeptides to access the cell interior (FIG. 9A). To quantify the relativeintracellular PCP delivery efficiency, HeLa cells were treated with eachpeptide and analyzed by fluorescence activated cell sorting (FIG. 9B).cFΦR₄-PCP was most efficiently internalized by the HeLa cells, with amean fluorescence intensity (MFI) of 3510 arbitrary units (AU), whereasR₉-PCP, Antp-PCP, Tat-PCP, and untagged PCP produced MFI values of 960,400, 290, and 30 AU, respectively (FIG. 9C). Again, when cells weretreated with cFΦR₄-PCP in the presence of sodium pervanadate, the amountof CAP fluorescence was reduced to near background levels (70 AU). Thus,cFΦR₄ is capable of delivering peptidyl cargos of varyingphysicochemical properties into the cytoplasm with efficiencies3.7-12-fold higher than R₉, Antp, and Tat.

TABLE 8 Kinetic Activities (k_(cat)/K_(M), M⁻¹ s⁻¹) of Recombinant PTPsagainst pCAP-Containing Peptides^(a) PTP cFΦR₄-PCP Tat-PCP R₉-PCPAntp-PCP PTP1B 37100 13800 14700 17400 TCPTP 2780 560 457 970 SHP2 74002290 248 2210 CD45 35100 21800 2940 22300 VHR 2460 1460 6240 2030^(a)k_(cat)/K_(M) was measured as previously described (Ren, L et al.Biochemistry, 2011, 50, 2339-2356).

Intracellular Delivery of Cyclic Peptides.

In recent years, there has been much interest in cyclic peptides astherapeutic agents and biomedical research tools (Driggers, E M et al.Nat. Rev. Drug Discov., 2008, 7, 608-624; Marsault, E and Peterson, M L.J. Med. Chem., 2011, 54, 1961-2004). For example, cyclic peptides areeffective for inhibition of protein-protein interactions (Lian, W et al.J. Am. Chem. Soc., 2013, 135, 11990-11995; Liu, T et al. ACS Comb. Sci.,2011, 13, 537-546; Dewan, V et al. ACS Chem. Biol., 2012, 7, 761-769;Wu, X et al. Med. Chem. Commun., 2013, 4, 378-382), which arechallenging targets for conventional small molecules. A major obstaclein developing cyclic peptide therapeutics is that they are generallyimpermeable to the cell membrane (Kwon, Y U and Kodadek, T. Chem. Biol.,2007, 14, 671-677; Rezai, T et al. J. Am. Chem. Soc., 2006, 128,2510-2511; Chatterjee, J et al. Acc. Chem. Res., 2008, 41, 1331-1342).The attempt to deliver cyclic peptides by cFΦR₄ by the endocyclic methodhad only limited success; increase in the cargo size from 1 to 7residues led to progressively poorer cellular uptake, likely because thelarger, more flexible rings bind more poorly to the cell membrane (Qian,Z et al. ACS Chem. Biol., 2013, 8, 423-431). To overcome thislimitation, a bicyclic peptide system was explored, in which one ringcontains a CPP motif (e.g., FΦR₄) while the other ring consists ofpeptide sequences specific for the desired targets (FIG. 1C). Thebicyclic system should in principle be able to accommodate cargos of anysize, because the cargo does not change the structure of the CPP ringand should have less impact on its delivery efficiency. The additionalrigidity of a bicyclic structure should also improve its metabolicstability as well as the target-binding affinity and specificity. Thebicyclic peptides were readily synthesized by forming three amide bondsbetween a trimesoyl scaffold and three amino groups on the correspondinglinear peptide (i.e., the N-terminal amine, the side chain of aC-terminal diaminopropionic acid (Dap), and the side chain of a lysine(or ornithine, Dap) imbedded in between the CPP and target-bindingmotifs) (Lian, W et al. J. Am. Chem. Soc., 2013, 135, 11990-11995). Totest the validity of this approach, FΦR₄ was chosen in the C-terminalring as the CPP moiety and peptides of different lengths and charges(AAAAA, AAAAAAA, RARAR, or DADAD) were chosen as cargo (Table 8,compounds 13-16). For comparison, two monocyclic peptides containingFΦR₄ as transporter and peptides A₅ and A₇ as cargos (Table 8, compounds17 and 18) were also prepared. All of the peptides were labeled at aC-terminal lysine side chain with rhodamine B (FIG. 2) and theirinternalization into HEK293 cells was examined by live-cell confocalmicroscopy. Treatment of cells with 5 μM peptide for 2 h resulted inefficient internalization of all six peptides (FIG. 10), although FACSanalysis indicated that the uptake of bicyclo(FΦR₄-A₅)^(Rho) was ˜3-foldmore efficient than the corresponding monocyclic peptide (compound 17).The intracellular distribution of the internalized peptides was quitedifferent between the bicyclic and monocyclic peptides. While the fourbicyclic peptides showed evidence for their presence in both thecytoplasm/nucleus (as indicated by the diffuse rhodamine fluorescence)and the endosomes (as indicated by the fluorescence puncta), themonocyclic peptides exhibited predominantly punctuate fluorescence thatoverlapped with that of the endocytic marker dextran^(FITC). In allcases, the endocytic marker displayed only punctuate fluorescence,indicating that the endosomes were intact in the cells treated with thepeptides. These results indicate that the increased structural rigidityof the bicyclic peptides facilitates both the initial uptake byendocytosis and endosomal release, presumably because of their improvedbinding to the plasma and endosomal membranes. The bicyclic system mayprovide a general strategy for intracellular delivery of cyclic andbicyclic peptides.

Intracellular Delivery of Protein Cargos.

To test whether cFΦR₄ is capable of transporting full-length proteinsinto mammalian cells, GFP was attached to the N-terminus of cFΦR₄through a disulfide bond (FIG. 11A and FIG. 3). GFP was chosen becauseof its intrinsic fluorescence. The disulfide exchange reaction is highlyspecific, efficient, and reversible; upon entering the cytoplasm, theCPP-S-S-protein conjugate can be rapidly reduced to release the nativeprotein. Although cFΦR₄ can be directly attached to a native orengineered surface cysteine residue(s) on a cargo protein, a GFP variantcontaining a 12-amino acid ybbR tag at its N-terminus was used andphosphopantetheinyl transferase Sfp was used to enzymatically attachcFΦR₄ to the ybbR tag (Yin, J et al. Proc. Natl. Acad. Sci. U.S.A.,2005, 102, 15815-15820). This permitted the attachment of a single cFΦR₄unit to GFP in a site-specific manner. For comparison, a Tat-S-S-GFPconjugate was generated in the same manner. Incubation of HEK293 cellsin the presence of 1 μM cFΦR₄—S-S-GFP resulted in accumulation of greenfluorescence inside the cells (FIG. 11B). The fluorescence signal wasdiffuse and present throughout the entire cell volume, but with higherconcentrations in the nucleus. Some of the cells contained small spotsof intense green fluorescence (indicated by arrows in FIG. 11B), whichmay represent endosomally sequestered cFΦR₄-S-S-GFP or aggregated GFPinside the cell. The untagged GFP was unable to enter cells, whereasTat-S-S-GFP entered cells less efficiently than cFΦR₄-S-S-GFP (FIG.11B); FACS analysis of HaLa cells treated with 1 μM protein revealed a5.5-fold higher total intracellular fluorescence for the latter. Thefluorescence puncta in the cell periphery as well as lack of anydetectable fluorescence in the nuclear region of Tat-S-S-GFP treatedcells indicate that Tat-S-S-GFP is mostly entrapped in the endosomes, inagreement with previous reports (Kaplan, I M et al. J. ControlledRelease, 2005, 102, 247-253). Thus, with a protein as cargo, cFΦR₄ alsohas higher efficiency than Tat with regard to both initial uptake andendosomal escape.

To demonstrate the generality of cFΦR₄ for protein delivery, afunctional enzyme, the catalytic domain of PTP1B (amino acids 1-321),was chosen to be delivered into the cell interior. To show that anon-cleavable linkage is also compatible with the delivery method, cFΦR₄was conjugated to ybbR-tagged PTP1B via a thioether bond (cFΦR₄-PTP1B)(FIG. 4). In vitro assay using p-nitrophenyl phosphate as substrateshowed that addition of the cFΦR₄ tag does not affect the catalyticactivity of PTP1B (Table 9). NIH 3T3 cells were incubated for 2 h in thepresence of untagged PTP1B or cFΦR₄-PTP1B and their global pY proteinlevels were analyzed by anti-pY western blotting (FIG. 12A). Treatmentof the cells with cFΦR₄-PTP1B, but not untagged PTP1B, resulted inconcentration-dependent decrease in pY levels of most, but not all,proteins. The total cellular protein levels, as detected by Coomassieblue staining, were unchanged (FIG. 12B), indicating that the observeddecrease in pY levels was due to dephosphorylation of the pY proteins bycFΦR₄-PTP1B and/or secondary effects caused by the introduction ofcFΦR₄-PTP1B (e.g., inactivation of cellular protein tyrosine kinases).Interestingly, different proteins exhibited varying dephosphorylationkinetics. Several proteins in the 150-200 kD range were completelydephosphorylated upon the addition of 62 nM cFΦR₄-PTP1B, whereasproteins of ˜80 kD remained phosphorylated at 500 nM cFΦR₄-PTP1B. Thechanges in the pY pattern are consistent with the broad substratespecificity of PTP1B (Ren, L et al. Biochemistry, 2011, 50, 2339-2356)and very similar to that caused by overexpression of PTP1B inside thecytosol of mammalian cells (LaMontagne Jr., K R et al. Proc. Natl. Acad.Sci. U.S.A., 1998, 95, 14094-14099). These results indicate that cFΦR₄can deliver PTP1B into the interior of NIH 3T3 cells in thecatalytically active form and to sufficient levels to perturb the cellsignaling process. cFΦR₄ thus provides a tool for introducing otherfunctional proteins, especially proteins that cannot be geneticallyexpressed (e.g., toxic and chemically modified proteins), into mammaliancells in order to study their cellular functions.

TABLE 9 Kinetic Activities (k_(cat)/K_(M), M⁻¹ s⁻¹) of PTP1B andcFΦR₄-PTP1B against pNPP^(a) enzyme k_(cat)/K_(M) (M⁻¹ s⁻¹) PTP1B 1340cFΦR₄-PTP1B 1600 ^(a)pNPP = p-nitrophenyl phosphate; k_(cat)/K_(M) wasmeasured as previously described (Ren, L et al. Biochemistry, 2011, 50,2339-2356).

Stability and Cytotoxicity of cFΦR₄.

The relative stability of cFΦR₄, R₉, Tat, and Antp (Table 8, compounds19-22) against proteolytic degradation was determined by incubating theCPPs in 25% human serum at 37° C. and following the disappearance of thefull-length peptides by reversed-phase HPLC. The cationictryptophan-containing peptide, Antp, was least stable among the fourCPPs; it was degraded at a half-life of <20 min and was completelydigested after 2 h (FIG. 13A). R9 and Tat were slightly more stable thanAntp, having half-lives of ˜30 min. In contrast, cFΦR₄ was remarkablystable against serum proteases. There was less than 10% degradationafter 6 h of incubation; after 24 h of incubation in the serum, >70% ofcFΦR₄ remained intact. Numerous other studies have also demonstratedthat cyclization of peptides increases their proteolytic stabilities(Nguyen, L T et al. PLoS One, 2010, 5, e12684). The potentialcytotoxicity of cFΦR₄ was assessed by MTT assays with five differenthuman cell lines (HEK293, MCF-7, A549, H1650, and H1299). After 24 or 48h of incubation with up to 50 μM cFΦR₄, there was no significant growthinhibition for any of the cell lines (FIG. 13B and FIG. 14). After 72 h,a slight growth inhibition (up to 20%) was observed at 50 μM (FIG. 14).Thus, cFΦR₄ is relatively nontoxic to mammalian cells.

In this study, it was demonstrated that cFΦR₄ can be effective forexocyclic delivery of small-molecule, peptide, and protein cargos intothe cytoplasm and nucleus of mammalian cells. By using a pCAP-containingpeptide as cargo/reporter, it was shown that cFΦR₄ can be 3.7-12-foldmore efficient than R9, Tat, and Antp for cytoplasmic cargo delivery,making cFΦR₄ one of the most active CPPs known to date. Althoughmodification of polybasic CPPs such as addition of hydrophobic acylgroups has previously been reported to enhance cellular uptake by asimilar magnitude (Pham, W et al. Chembiochem, 2004, 5, 1148-1151),these previous studies have not established whether the enhanced uptaketranslates into a similar increase in the cytoplasmic CPP concentration.The pCAP-based reporter system described herein can provide a simple,robust method to quantitatively assess the cytoplasmic deliveryefficiency of other CPPs. Several lines of evidence indicate that cFΦR₄can enter cells through multiple endocytic mechanisms, including itsfailure to enter cells at 4° C. or in the presence of sodium azide,partial overlap between the fluorescence puncta of cFΦR₄ ^(Rho) and thefluid phase endocytic marker dextran^(FITC), colocalization of cFΦR₄^(Rho) and endosomal proteins Rab5 and Rab7, and decreased cFΦR₄ ^(Dex)uptake upon administration of endocytic inhibitors. The minimal effectof the PI3K inhibitor wortmannin and the Rab5 Q79L mutation on thecytoplasmic delivery of cFΦR₄, in addition to the strong colocalizationobserved between cFΦR₄ and Rab5⁺ endosomes, suggest that cFΦR₄ canescape from early endosomes (FIG. 15). In comparison, Tat has beendemonstrated to enter cells through endocytosis and release from lateendosomes, while R9 escapes endosomes prior to Rab7 recruitment(Appelbaum, J S et al. Chem. Biol., 2012, 19, 819-830). Early endosomalrelease can offer advantages, especially for peptide and protein cargos,since it can minimize cargo degradation by late endosomal and lysosomalproteases and denaturation caused by acidification during endosomalmaturation. Indeed, both GFP and PTP1B delivered into the cytoplasm bycFΦR₄ were in their folded, active forms, as evidenced by the greenfluorescence and the ability to dephosphorylate intracellular pYproteins, respectively. Additionally, due to its more rigid structure,cFΦR₄ can be more stable against proteolytic degradation than linearpeptides, and due to its smaller size, cFΦR₄ can be less expensive tosynthesize and potentially less likely to interfere with the cargofunction. These properties can make cFΦR₄ a useful transporter forcytosolic delivery of small-molecule to protein cargos. Direct proteindelivery can provide a useful research tool, e.g., for studying thecellular function of a protein, as it can offer improved temporalcontrol over DNA transfection and subsequent gene expression and canallow delivery of chemically modified proteins and proteins whoseoverexpression can cause toxicity. The ability of cFΦR₄ to escape fromearly endosomes and its simple structure can also provide an excellentsystem for elucidating the mechanism of endosomal escape and the factorsthat influence the escape efficiency.

Example 2

Cyclization of peptide ligands can be effective for improving theirstability against proteolytic degradation and in some cases their cellpermeability. However, this strategy is not compatible with proteinsthat recognize peptide ligands in the extended conformations (e.g.,β-strand and α-helix). In this work, a general strategy forintracellular delivery of linear peptide ligands was developed, byfusing them with an amphipathic sequence motif (e.g., RRRRΦF, where Φ isL-naphthylalanine) and cyclizing the resulting conjugate through adisulfide bond. The cyclized peptides can have enhanced proteolyticstability and membrane permeability; upon entering the cytoplasm/nucleusof a cell, the disulfide bond can be cleaved by the reducingintracellular environment to release the linear, biologically activepeptide. This strategy was applied to generate cell permeable peptidesas caspase substrates and inhibitors against the CAL PDZ domain forpotential treatment of cystic fibrosis.

The applicability of linear peptides as drugs is often limited by theirsusceptibility to proteolytic cleavage and poor membrane permeability.Cyclization of peptides can be effective for improving their proteolyticstability (Nguyen, L T et al. PLoS One, 2010, 5, e12684). Moreover, itwas recently reported that cyclization of certain amphipathic peptides(e.g., FΦRRRR, where Φ is L-2-naphthylalanine) can render them cellpermeable through an active transport mechanism (Qian, Z et al. ACSChem. Biol. 2013, 8, 423). Biologically active cyclic peptides can bedelivered into the cytoplasm and nucleus of mammalian cells byincorporating into them these short sequence motifs (Qian, Z et al. ACSChem. Biol. 2013, 8, 423). However, in many circumstances, binding to amolecular target (e.g., PDZ (Doyle, D A et al. Cell 1996, 85, 1067;Morais Cabral, J H et al., Nature 1996, 382, 649) and BIR domains (Wu, Get al. Nature 2000, 408, 1008)) can require that the peptidyl ligandexist in its extended conformation (e.g., α-helix and β-strand) andcyclization may interfere with target binding. Herein, a potentiallygeneral strategy for delivering linear peptide ligands into mammaliancells through reversible, disulfide bond-mediated cyclization isexamined. When present in the oxidizing extracellular environment, thepeptides can exist as macrocycles, which can have enhanced stabilityagainst proteolysis and cell permeability. Upon entering the cell (i.e.,cytoplasm and/or nucleus), the disulfide bond can be reduced by theintracellular thiols to produce the linear, biologically active peptides(FIG. 16) (Cascales, L et al. J. Biol Chem. 2011, 286, 36932; Jha, D etal. Bioconj Chem. 2011, 22, 319).

Materials.

Reagents for peptide synthesis were purchased from Advanced ChemTech(Louisville, Ky.), NovaBiochem (La Jolla, Calif.), or Anaspec (San Jose,Calif.). Rink resin LS (100-200 mesh, 0.2 mmol/g) was purchased fromAdvanced ChemTech. Dextrane^(Rho), trypsin and α-chymotrypsin werepurchased from Sigma-Aldrich (St. Louis, Mo.). Cell culture media, fetalbovine serum, penicillin-streptomycin, 0.25% trypsin-EDTA, and DPBS werepurchased from Invitrogen (Carlsbad, Calif.). Nuclear staining dye DRAQ5was purchased from Thermo Scientific (Rockford, Ill.). Caspase-3, Human,recombinant protein was purchased from EMD Chemicals (San Diego,Calif.).

Peptide Synthesis.

Most peptides were synthesized on Rink Resin LS (0.2 mmol/g) usingstandard Fmoc chemistry. The typical coupling reaction contained 5 equivof Fmoc-amino acid, 5 equiv of2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) and 10 equiv of diisopropylethylamine (DIPEA)and was allowed to proceed with mixing for 75 min. The peptides weredeprotected and released from the resin by treatment with92.5:2.5:2.5:2.5 (v/v) trifluoroacetic acid(TFA)/water/phenol/triisopropylsilane (TIPS) for 2 h. The peptides weretriturated with cold ethyl ether (3×) and purified by reversed-phaseHPLC equipped with a C18 column. Peptide labeling with fluoresceinisothiocyanate (FITC) was performed by dissolving the purified peptides(˜1 mg each) in 300 μL of 1:1:1 DMSO/DMF/150 mM sodium bicarbonate (pH8.5) and mixing with 10 μL of FITC in DMSO (100 mg/mL). After 20 min atroom temperature, the reaction mixture was subjected to reversed-phaseHPLC on a C₁₈ column to isolate the FITC-labeled peptide.

To generate disulfide bond mediated cyclic peptides, the3,3′-dithiodipropionic acid (10 equiv) were coupled on the N-terminalusing 10 equiv N,N′-Diisopropylcarbodiimide (DIC) and 0.1 equiv4-(dimethylamino)pyridine (DMAP) in anhydrous DCM for 2 h after theremoval of the N-terminal Fmoc protection group by treatment with 20%(v/v) piperidine in DMF. The resin then was incubated in 20%β-mercaptoethanol in DMF for 2 h twice to expose the free thiol.Triturated crude linear peptides were incubated in 5% DMSO in pH 7.4 PBSbuffer overnight (Tam, J P et al. J. Am. Chem. Soc. 1991, 113, 6657),followed by trituration and HPLC purification as described above (Tam, JP et al. J. Am. Chem. Soc. 1991, 113, 6657).

To produce thioether mediated cyclic peptides, 4-bromobutyric acid (10equiv) was coupled on the N-terminal using 10 equiv DIC and 0.1 equivDMAP in anhydrous DCM for 2 h after the removal of the N-terminal Fmocprotection group by treatment with 20% (v/v) piperidine in DMF. The4-methoxytrityl (Mmt) protection group on the L-cysteine side chain wasselectively removed using 1% trifluoroacetic acid (TFA) in DCM.Thioether formation was conducted by incubating the resin in 1% DIPEA inDMF under nitrogen protection overnight. The cyclized peptide was thentriturated and purified as described above (Roberts, K D et al.Tetrahedron Lett. 1998, 39, 8357).

Fmoc-Asp(Wang-resin)-AMC (AMC=7-amino-4-methylcoumarin) (NovaBiochem)was used as a solid support to synthesize fluorogenic caspasesubstrates. Standard Fmoc chemistry was employed to synthesize thepeptide on solid phase. These peptides were released from the resin bythe treatment with 95:2.5:2.5 (v/v) TFA/phenol/water for 2 h (Maly, D Jet al. J. Org. Chem. 2002, 67, 910).

Cell Culture.

HeLa cells were maintained in medium consisting of DMEM, 10% fetalbovine serum (FBS) and 1% penicillin/streptomycin. Jurkat cells weremaintained in medium consisting of RPMI-1640, 10% FBS and 1%penicillin/streptomycin. The bronchial epithelial CFBE cell line,homozygous for the ΔF508-CFTR mutation, was maintained in DMEMcontaining L-glutamine supplemented with 10% FBS and 1%penicillin/streptomycin. The tissue culture plates were coated usinghuman fibronectin (1 mg/ml), collagen I bovine (3 mg/ml), and bovineserum albumin (1 mg/ml) Cells were cultured in a humidified incubator at37° C. with 5% CO₂.

Confocal Microscopy.

To detect peptide internalization, 1 mL of HeLa cell suspension (5×10⁴cells) was seeded in a 35 mm glass-bottomed microwell dish (MatTek) andcultured overnight. Cells were gently washed with DPBS twice and treatedwith FITC labeled peptides (5 μM) and dextran^(Rho) (0.5 mg mL⁻¹) inphenol-red free DMEM containing 1% serum at 37° C. for 1 h in thepresence of 5% CO₂. After removal of the medium, the cells were gentlywashed with DPBS twice and incubated with 5 μM DRAQS in DPBS for 10 min.The cells were again washed with DPBS twice and imaged on a VisitechInfinity 3 Hawk 2D-array live cell imaging confocal microscope. Imageswere captured under the same parameters and adjusted under the samesetting using MetaMorph (Molecular Devices).

Flow Cytometry.

HeLa cells were cultured in six-well plates (5×10⁵ cells per well) for24 h. On the day of experiment, the cells were incubated with 5 μM FITClabeled peptide in clear DMEM with 1% FBS at 37° C. for 2 h. The cellswere washed with DPBS, detached from plate with 0.25% trypsin, dilutedinto clear DMEM containing 10% FBS, pelleted at 250 g for 5 min, washedonce with DPBS and resuspended in DPBS containing 1% bovine serumalbumin, and analyzed on a BD FACS Aria flow cytometer. Data wereanalyzed with Flowjo software (Tree Star).

To quantify the delivery efficiencies of PCP-conjugated peptides, HeLacells were cultured in six-well plates (5×10⁵ cells per well) for 24 h.On the day of experiment, the cells were incubated with 5 μMpCAP-containing peptide in clear DMEM with 1% FBS at 37° C. for 2 h. Thecells were washed with DPBS containing 1 mM sodium pervanadate, detachedfrom plate with 0.25% trypsin, suspended in DPBS containing 1% bovineserum albumin, and analyzed on a BD FACS Aria flow cytometer withexcitation at 355 nm.

Peptide Proteolysis Stability Assay.

The stability tests were carried out by slightly modifying a previouslyreported procedure (Frackenpohl, J et al. Chembiochem 2001, 2, 445). 24μL of 1.5 mM peptide solution was incubated at 37° C. with 30 μL 50 μMof α-chymotrypsin and 30 μL 50 μM of trypsin in 200 μL of working buffer(50 mM Tris-HCl, pH 8.0, NaCl (100 mM), CaCl₂ (10 mM)). At various timepoints (0-12 h), 40 μL aliquots were withdrawn and mixed with 40 μL of15% trichloroacetic acid and incubated at 4° C. overnight. The finalmixture was centrifuged at 15,000 rpm for 10 min in a microcentrifuge,and the supernatant was analyzed by reversed-phase HPLC equipped with aC18 column (Waters). The amount of remaining peptide (%) was determinedby integrating the area underneath the peptide peak (monitored at 214nm) and compared with that of control reaction (no proteases).

In Cellulo Fluorimetric Assay.

100 μL of Jurkat cell suspension (5×10⁵ cells/mL) was seeded in 96-wellplate one hour prior to the experiment. Ten μL of staurosporine stocksolution (10 μM) was added into half of the wells to induce apoptosis,while 10 μL of media was added to the other wells. After 1 h incubation,caspase-3 fluorogenic substrates were added to the cells to a finalconcentration of 5 μM. The fluorescence of the released coumarin wasmeasured on the Spectramax M5 plate reader with excitation and emissionwavelengths at 360 and 440 nm at various times points (0-6 h). Thefluorescence unit (FU) increases between induced and uninduced cellswere plotted against the time to present caspase-3 activities measuredusing various fluorogenic substrates in living cell in real-time. Threeindependent sets of experiments, each performed in triplicate, wereconducted.

In Vitro Fluorimetric Assay.

0.5 μL (100 U/μL) caspase-3 enzyme was first incubated with 90 μL ofreaction buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM DTT) for 30 minin 96-well plate. Fluorogenic substrates (10 μL, 100 μM) were mixed intothe above solutions to start the reactions, and the plate was measuredon a Spectramax M5 plate reader (Ex=360 nm, Em=440 nm) (MolecularDevices). Fluorescence units (FU) increase at one-minute intervals wascorrelated to the release of Amc due to protease activity. The ΔFU/minwas calculated from the linear portion of the reaction curve. Reportedvalues are averages of three trials with the standard deviationindicated.

Fluorescence Anisotropy.

The full fluorescence anisotropy (FA) titration experiment was performedby incubating 100 nM fluorophore-labeled peptidyl ligands with varyingconcentrations (0-6 μM) of CAL-PDZ (Cushing, P R et al. Biochemistry2008, 47, 10084) in FA buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mMglutathione, 0.1% (w/v) bovine serum albumin) for 2 h at roomtemperature. The FA values were measured on a Molecular DevicesSpectramax M5 spectrofluorimeter, with excitation and emissionwavelengths at 485 nm and 525 nm, respectively. Equilibrium dissociationconstants (K_(D)) were determined by plotting the fluorescenceanisotropy values as a function of CAL-PDZ concentration. The titrationcurves were fitted to the following equation, which assumes a 1:1binding stoichiometry

$Y = \frac{\left( {A_{m\; i\; n} + {\left( {{A_{{ma}\; x} \times \frac{Q_{b}}{Q_{f}}} - A_{m\; i\; n}} \right)\left( \frac{\begin{matrix}{\left( {L + x + K_{D}} \right) -} \\\sqrt{\left( {\left( {L + x + K_{D}} \right)^{2} - {4{Lx}}} \right)}\end{matrix}}{2L} \right)}} \right)}{\left( {1 + {\left( {\frac{Q_{b}}{Q_{f}} - 1} \right)\left( \frac{\left( {L + x + K_{D}} \right) - \sqrt{\left( {\left( {L + x + K_{D}} \right)^{2} - {4{Lx}}} \right)}}{2L} \right)}} \right)}$

where Y is the measured anisotropy at a given CAL-PDZ concentration x; Lis the bicyclic peptide concentration; Q_(b)/Q_(f) is the correctionfact for dye-protein interaction; A_(max) is the maximum anisotropy whenall the peptides are bound to CAP-PDZ, while A_(min) is the minimumanisotropy when all the peptides are free.

Immunofluorescent Staining.

Briefly, the bronchial epithelial CFBE cells, homozygous for theDF508-CFTR mutation, were treated with 10 mM Corr-4a in the presence andabsence of 50 μM unlabeled peptide 8. After the treatments, cells werefixed in cold methanol for 20 min. The slides were then incubated in 1%BSA/PBS for 10 min, followed by incubation at 37° C. for 1 h with mouseanti-human monoclonal CFTR antibody (R&D Systems). Thereafter, theslides were incubated at 37° C. for 45 min with Alexa Fluor®488-conjugated anti-mouse IgG2a secondary antibody. Cells werevisualized on a Leica TCS SP2 AOBS confocal laser scanning microscope.All measurements were conducted in a double-blinded manner by twoindependent investigators.

SPQ Intracellular Chloride Concentration Assay.

A SPQ (6-Methoxy-N-(3-sulfopropyl)quinolinium) assay was utilized toestimate the transport activity of ΔF508-CFTR activity in CFBE cells, asthe fluorescence of SPQ is negatively correlated with increasingconcentration of intracellular chloride (Illsley, N P and Verkman, A S.Biochemistry 1987, 26, 1215). CFBE cells were grown on 96-well plate,which was pre-coated with 1 mg/ml human fibronectin, 3 mg/ml collagen Ibovine, and 1 mg/ml bovine serum albumin, using DMEM media supplementedwith L-glutamine and 10% FBS. Cells were first treated in the presenceor absence of 20 μM CFTR corrector VX809 (Van Goor, F et al. Proc. Natl.Acad. Sci. U.S.A. 2011, 108, 18843) for 24 h and 50 μM CAL-PDZ domaininhibitors for 1 h. Cells were then loaded with SPQ using hypotonicshock at 37° C. for 15 min with 10 mM SPQ containing 1:1 (v/v)Opti-MEM/water solution. The cells were then washed and incubated twicefor 10 min with fluorescence quenching NaI buffer (130 mM NaI, 5 mMKNO₃, 2.5 mM Ca(NO₃)₂, 2.5 mM Mg(NO₃)₂, 10 mM D-glucose, 10 mMN-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic) acid (HEPES, pH7.4)). Subsequently, the cells were switched to a dequenching isotonicNaNO₃ buffer (identical to NaI buffer except that 130 mM NaI wasreplaced with 130 mM NaNO₃) with a CFTR activation cocktail (10 μMforskolin and 50 μM genistein). Fluorescence non-specific toCFTR-mediated iodide efflux was measured by incubating the cells withthe activation cocktail and the CFTR specific inhibitor GlyH101 (10 μM).The effects of CAL-PDZ inhibitors were evaluated by the fluorescenceincreasing rate above the basal level. The fluorescence of dequenchedSPQ was measured using the plate reader VICTOR X3 (Perkin Elmer) withexcitation wavelength at 350 nm and DAPI emission filter. The data waspresented as mean±standard deviation from at least three individualexperiments.

A homodectic amphipathic cyclic peptide, cyclo(FΦRRRRQ) (cFΦR₄), hasbeen reported as a highly active cell-penetrating peptide (CPP) whichcan enter the cytoplasm of mammalian cells through endocytosis andendosomal escape (Qian, Z et al. ACS Chem. Biol. 2013, 8, 423). To testthe validity of the reversible cyclization strategy, aN-3-mercaptopropionyl-FΦRRRRCK-NH₂ peptide was synthesized and thencyclized by forming an intramolecular disulfide bond (FIG. 17; Table 10,peptide 1). A linear peptide of the same sequence (Table 10, peptide 2)was also synthesized by replacing the N-terminal 3-mercaptopropionylgroup with a butyryl group and the C-terminal cysteine with2-aminobutyric acid (Abu or U). Both peptides were labeled at aC-terminal lysine residue with fluorescein isothiocyanate (FITC) andtheir cellular uptake was assessed by live-cell confocal microscopy andflow cytometry. HeLa cells treated with the cyclic peptide (5 μM) showedstrong, diffuse green fluorescence throughout the entire cell volume,whereas the endocytosis marker, rhodamine-labeled dextran(dextran^(Rho)), exhibited only punctuate fluorescence in thecytoplasmic region (FIG. 18A). The nearly uniform distribution of FITCfluorescence in both cytoplasmic and nuclear regions suggests that thecyclic peptide was efficiently internalized by HeLa cells and like theparent cyclic peptide, cFΦR₄, was able to efficiently escape from theendosome. In contrast, cells treated with the linear control peptideshowed much weaker intracellular fluorescence under the same imagingcondition. Quantitation of the total intracellular fluorescence byfluorescence-activated cell sorting (FACS) gave mean fluorescenceintensity (MFI) of 27, 100, 5530, and 1200 arbitrary units (AU), forcells treated with the disulfide cyclized peptide, linear peptide, andFITC alone, respectively (FIG. 18B). A highly negatively chargedpentapeptide, Asp-Glu-pCAP-Leu-Ile (PCP, where pCAP is phosphocoumarylaminopropionic acid), was also used as cargo and attached to peptides 1and 2 through a polyethyleneglycol linker (FIG. 17). pCAP isnon-fluorescent but, when delivered into the mammalian cytoplasm,undergoes rapid dephosphorylation to generate a fluorescent product,coumaryl aminopropionic acid (CAP) (Stanford, S M et al. Proc. Natl.Acad. Sci. U.S.A. 2012, 109, 13972). The pCAP assay therefore provides aquantitative assessment of the cytoplasmic/nuclear concentrations ofdifferent CPPs (Qian, Z et al. ACS Chem. Biol. 2013, 8, 423). FACSanalysis of HeLa cells treated with 5 μM peptide 1-PCP and peptide 2-PCPgave MFI values of 3020 and 700, respectively (FIG. 19). Thus, the aboveresults indicate that cyclization of FΦRRRR through a disulfide bond canhave a similar effect to the N-to-C cyclization and can increase itscellular uptake efficiency by ˜5-fold (Qian, Z et al. ACS Chem. Biol.2013, 8, 423). In addition, cyclization by disulfide bond formation canenhance the proteolytic resistance of the peptide. Incubation of peptide1 with a protease cocktail for 12 h resulted in <50% degradation,whereas the linear peptide 2 was degraded with a half-life of ˜20 minunder the same condition (FIG. 20).

TABLE 10 Sequences of peptides. SEQ ID Peptide NO ID PeptideSequence^(a) 123  1

124  2 CH₃CH₂CH₂CO-FΦRRRRUK(FITC)-NH₂ 125  3 Ac-DMUD-Amc 126  4

127  5

128  6 CH₃CH₂CH₂CO⁻RRRRΦFDΩUD⁻A^(mc) 129  7 A^(c−)RRRRRRRRRDΩUD⁻A^(mc)130  8

131  9 FITC-URRRRFWQUTRV-OH 132 11

^(a)Amc, 7-amino-4-methylcourmarin; FITC, fluorescein isothiocyanate; Φ,L-2-naphthylalanine; Ω, norleucine; U, 2-aminobutyric acid.

To illustrate the utility of the reversible cyclization strategy, it wasused to deliver specific caspase substrates into cells and monitorintracellular caspase activities in real time (Riedl, S J and Shi, Y.Nat. Rev. Mol. Cell Biol. 2004, 5, 897). Although peptidyl coumarinderivatives have been widely used to detect caspase activities in vitro(Maly, D J et al. Chembiochem 2002, 3, 16), they are generally notsuitable for in vivo applications due to impermeability to the mammaliancell membrane. To generate a cell permeable caspase substrate, a caspase3/7 substrate, Ac-Asp-Nle-Abu-Asp-Amc (Thornberry, N A et al. J. Biol.Chem. 1997, 272, 17907) (Table 10, peptide 3, where Amc is7-amino-4-methylcoumarin and Nle is norleucine), was fused with the CPPmotif RRRRΦF. The fusion peptide was subsequently cyclized by theaddition of a 3-mercaptopropionyl group to its N-terminus, replacementof the C-terminal Abu with a cysteine, and formation of anintramolecular disulfide bond, to give cyclic peptide 4 (Table 10). Forcomparison, an isosteric but irreversibly cyclized peptide (Table 10,peptide 5) was synthesized by forming a thioether bond between anN-terminal bromobutyryl moiety and the C-terminal cysteine (FIG. 17). Alinear control peptide of the same sequence was also prepared asdescribed above (Table 10 peptide 6). Finally, the caspase 3/7 substratewas conjugated to nonaarginine (R9) to generate a positive controlpeptide (Table 10, peptide 7). In vitro kinetic analysis revealed thatfusion of the caspase 3/7 substrate to RRRRΦF and R9 decreased itsactivity by 53% and 72%, respectively, relative to peptide 3, whereascyclization by thioether formation rendered the peptide inactive towardrecombinant caspase 3 (Table 11). The activity of peptide 4 towardcaspase 3 could not be reliably determined because the caspase assayrequired a reducing environment, which would cleave the disulfide bond.Given the structural similarity between peptides 4 and 5, it can beassumed that peptide 4 in the cyclic form is also inactive towardcaspases, but has similar activity to peptide 6 after reductive cleavageof the disulfide bond.

TABLE 11 In vitro activity of various fluorogenic substrates againstrecombinant caspase-3 enzyme. Peptide ID ΔFU/min 3 159 ± 19 5 Nodetectable activity 6 74.7 ± 5.5 7 45.3 ± 6.5

Jurkat cells were pretreated with the kinase inhibitor staurosporin toinduce caspase activities and thus apoptosis (Belmokhtar, C A et al.Biochem. J. 1996, 315, 21). These cells were then incubated withpeptides 3-7 and the amount of Amc released was monitored at varioustime points (0-10 h). The impermeable caspase substrate (peptide 3)produced little fluorescence increase over the 10-h period (FIG. 21).Peptide 4 produced the fastest fluorescence increase, reaching 459fluorescence units (FU), followed by peptides 7 and 6. Peptide 5, whichis inactive toward caspase 3, also produced AMC in a time-dependentmanner, albeit at a much slower rate (99 FU). This slow rate of AMCrelease can be attributed to hydrolysis by other intracellular proteasesand peptidases. Consistent with this interpretation, pretreatment ofJurkat cells with a pancaspase inhibitor Z-VAD(OMe)-FMK (Slee, E A etal. Biochem J. 1996, 315, 21) followed by incubation with peptide 4released AMC at a rate that was similar to that of peptide 5 alone. Oneexplanation of the above observations is that both peptides 4 and 5 canenter the cell interior efficiently, but only peptide 4 can be convertedinto the linear caspase substrate inside the cells.

Many protein-protein interactions (PPIs) are mediated by protein domainsbinding short peptides in their extended conformations (e.g., α-helixand β-strand) (Pawson, T and Nash, P. Science 2003, 300, 445). Forexample, the PDZ domain is a common structural domain of 80-90 aminoacids found in the signaling proteins of bacteria to man (Doyle, D A etal. Cell 1996, 85, 1067; Morais Cabral, J H et al., Nature 1996, 382,649; Lee, H J and Zheng, J J. Cell Commun. Signal. 2010, 8, 8). PDZdomains recognize specific sequences at the C-termini of their bindingpartners and the bound peptide ligands are in their extended β-strandconformation (Doyle, D A et al. Cell 1996, 85, 1067; Songyang, Z et al.Science 1997, 275, 73). It was recently reported that the activity ofcystic fibrosis membrane conductance regulator (CFTR), a chloride ionchannel protein mutated in cystic fibrosis (CF) patients, is negativelyregulated by CFTR-associated ligand (CAL) through its PDZ domain(CAL-PDZ) (Wolde, M et al. J. Biol. Chem. 2007, 282, 8099). Inhibitionof the CFTR/CAL-PDZ interaction was shown to improve the activity ofΔPhe508-CFTR, the most common form of CFTR mutation (Cheng, S H et al.Cell 1990, 63, 827; Kerem, B S et al. Science 1989, 245, 1073), byreducing its proteasome-mediated degradation (Cushing, P R et al. Angew.Chem. Int. Ed. 2010, 49, 9907). Previous library screening and rationaldesign have identified several peptidyl inhibitors of the CAL-PDZ domainof moderate potencies (K_(D) values in the high nM to low μM range)(Cushing, P R et al. Angew. Chem. Int. Ed. 2010, 49, 9907; Roberts, K Eet al. PLos Comput. Biol. 2008, 8, e1002477; Kundu, R et al. Angew.Chem. Int. Ed. 2012, 51, 7217-7220). However, none of the peptideinhibitors were cell permeable, limiting their therapeutic potential.

Starting with a hexapeptide ligand for the CAL-PDZ domain, WQVTRV(Roberts, K E et al. PLos Comput. Biol. 2008, 8, e1002477), adisulfide-mediated cyclic peptide was designed by adding the sequenceCRRRRF to its N-terminus and replacing the Val at the −3 position with acysteine (Table 10, peptide 8). Thus, in peptide 8, the tryptophanresidue at the −5 position was designed to serve the dual function ofPDZ binding and membrane translocation. To facilitate affinitymeasurements and quantitation of its cellular uptake, a FITC group wasadded to the N-terminus of peptide 8. FA analysis showed that in theabsence of a reducing agent, peptide 8 showed no detectable binding toCAL-PDZ domain (FIG. 22A). In the presence of 2 mMtris(carboxylethyl)phosphine, which can reduce the disulfide bond,peptide 8 bound to the CAL-PDZ domain with a K_(D) value of 489 nM.Peptide 8 was readily cell permeable; incubation of HeLa cells with 5 μMpeptide 8 for 2 h resulted in intense and diffuse fluorescencethroughout the entire cell (FIG. 22B).

As expected, peptide 8 is readily cell permeable (FIG. 25C). Bronchialepithelial CFBE cells, which are homozygous for the ΔF508-CFTR mutation,were treated with 10 μM Corr-4a in the presence and absence of 50 μMunlabeled peptide 8. Peptide 8, by inhibiting the function of CAL-PDZdomain, is expected to increase the amount of ΔF508-CFTR proteintransferred to the plasma membrane, whereas Corr-4a is a small moleculethat helps folding of ΔF508-CFTR protein delivered to the plasmamembrane. Immunostaining of untreated cells (FIG. 25D, panel I) showedthat most of the expressed ΔF508-CFTR was in the endoplasmic reticulumsurrounding the cell nucleus. In contrast, treatment of cells withCorr-4a and peptide 8 resulted in much greater amounts of the protein atthe cell surface (FIG. 25D panel II). Quantitation of the cellpopulation revealed that a small but significant percentage of cellshave wild-type like distribution of ΔF508-CFTR at the cell surface (FIG.25D). Finally, an SPQ assay was utilized to quantitate the ion channelactivity of ΔF508-CFTR CFBE cells untreated or treated with CTFR foldingcorrector VX809 and peptide 8. Again, VX809 and peptide 8 actedsynergistically to improve the function of the channel activity ofΔF508-CFTR (FIG. 25E).

Example 3

Cyclic peptides have great potential as therapeutic agents and researchtools but are generally impermeable to the cell membrane. Fusion of thecyclic peptides with a cyclic cell-penetrating peptide can producebicyclic peptides that can be cell permeable and can retain the abilityto recognize specific intracellular targets. Application of thisstrategy to protein tyrosine phosphatase 1B and peptidyl prolylcis-trans isomerase Pin1 resulted in potent, selective, proteolyticallystable, and biologically active inhibitors against the enzymes.

Cyclic peptides (and depsipeptides) exhibit a wide range of biologicalactivities (Pomilio, A B et al. Curr. Org. Chem. 2006, 10, 2075-2121).Several innovative methodologies have recently been developed tosynthesize cyclic peptides, either individually (Meutermans, W D F etal. J. Am. Chem. Soc. 1999, 121, 9790-9796; Schafmeister, C E et al. J.Am. Chem. Soc. 2000, 122, 5891-5892; Sun, Y et al. Org. Lett. 2001, 3,1681-1684; Kohli, R M et al. Nature 2002, 418, 658-661; Qin, C et al. J.Comb. Chem. 2004, 6, 398-406; Turner, R A et al. Org. Lett. 2007, 9,5011-5014; Hili, R et al. J. Am. Chem. Soc. 2010, 132, 2889-2891; Lee, Jet al. J. Am. Chem. Soc. 2009, 131, 2122-2124; Frost, J R et al.ChemBioChem 2013, 14, 147-160) or combinatorially (Eichler, J et al.Mol. Divers. 1996, 1, 233-240; Giebel, L B et al. Biochemistry 1995, 34,15430-15435; Scott, C P et al. Proc. Natl. Acad. Sci. USA 1999, 96,13638-13643; Millward, S W et al. I Am. Chem. Soc. 2005, 127,14142-14143; Sako, Y et al. J. Am. Chem. Soc. 2008, 130, 7232-7234.; Li,S et al. Chem. Commun. 2005, 581-583.; Joo, S H et al. J. Am. Chem. Soc.2006, 128, 13000-13009; Heinis, C et al. Nat. Chem. Biol. 2009, 5,502-507; Tse, B N et al. J. Am. Chem. Soc. 2008, 130, 15611-15626), andscreen them for biological activity. A particularly exciting applicationof cyclic peptides is the inhibition of protein-protein interactions(PPIs) (Leduc, A M et al. Proc. Natl. Acad. Sic. USA 2003, 100,11273-11278; Millward, S W et al. ACS Chem Biol 2007, 2, 625-634;Tavassoli, A et al. ACS Chem. Biol. 2008, 3, 757-764.; Wu, X et al. Med.Chem. Commun. 2013, 4, 378-382; Birts, C N et al. Chem. Sci. 2013, 4,3046-3057; Kawakami, T et al. ACS Chem. Biol. 2013, 8, 1205-1214; Lian,W et al. J. Am. Chem. Soc. 2013, 135, 11990-11995), which remainchallenging targets for conventional small molecules. However, a majorlimitation of cyclic peptides is that they are generally impermeable tothe cell membrane, precluding any application against intracellulartargets, which include most of the therapeutically relevant PPIs.Although formation of intramolecular hydrogen bonds (Rezai, T et al. J.Am. Chem. Soc. 2006, 128, 14073-14080) or N^(α)-methylation of thepeptide backbone (Chatterjee, J et al. Acc. Chem. Res. 2008, 41,1331-1342; White, T R et al. Nat. Chem. Biol. 2011, 7, 810-817) canimprove the membrane permeability of certain cyclic peptides,alternative strategies to increase the cell permeability of cyclicpeptides are clearly needed.

Protein-tyrosine phosphatase 1B (PTP1B) is a prototypical member of thePTP superfamily and plays numerous roles during eukaryotic cellsignaling. Because of its role in negatively regulating insulin andleptin receptor signaling, PTP1B is a valid target for treatment of typeII diabetes and obesity (Elchelby, M et al. Science 1999, 283,1544-1548; Zabolotny, J M et al. Dev. Cell 2002, 2, 489-495). A largenumber of PTP1B inhibitors have been reported (He, R et al. in NewTherapeutic Strategies for Type 2 Diabetes: Small Molecule Approaches.Ed. R. M. Jones, RSC Publishing 2012, pp 142), however, none of themhave succeeded in the clinic. Designing PTP inhibitors is challengingbecause most of the phosphotyrosine (pY) isosteres, such asdifluorophosphonomethyl phenylalanine (F₂Pmp) (Burke Jr., T R et al.Biochem. Biophys. Res. Commun. 1994, 204, 129-134), are impermeable tothe cell membrane. Additionally, because all PTPs share a similar activesite, achieving selectivity for a single PTP has been difficult. Herein,a potentially general approach to designing cell-permeable cyclicpeptidyl inhibitors against intracellular proteins such as PTP1B isreported.

Materials.

Fmoc-protected amino acids were purchased from Advanced ChemTech(Louisville, Ky.), Peptides International (Louisville, Ky.), or Aapptec(Louisville, Ky.). Fmoc-F2Pmp-OH was purchased from EMD Millipore(Darmstadt, Germany). Aminomethyl-ChemMatrix resin (0.66 mmol/g) wasfrom SJPC (Quebec, Canada). Rink resin LS (100-200 mesh, 0.2 mmol/g) andN-(9-fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu) were purchasedfrom Advanced ChemTech. O-Benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU),2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), 1-hydroxybenzotriazole hydrate (HOBt) werepurchased from Aapptec. Phenyl isothiocyanate in 1-mL sealed ampoules,fluorescein isothiocyanate (FITC), rhodamine B-labeled dextran(dextran^(Rho)) were purchased from Sigma-Aldrich. Cell culture media,fetal bovine serum (FBS), penicillin-streptomycin, 0.25% trypsin-EDTA,Dulbecco's phosphate-buffered saline (DPBS) (2.67 mM potassium chloride,1.47 mM potassium phosphate monobasic, 137 mM sodium chloride, 8.06 mMsodium phosphate dibasic.), and anti-phospho-IR/IGF1R antibody werepurchased from Invitrogen (Carlsbad, Calif.). Nuclear staining dye DRAQ5and anti-β-actin antibody were purchased from Thermo Scientific(Rockford, Ill.). Antibody 4G10 was purchased from Millipore (Temecula,Calif.). All solvents and other chemical reagents were obtained fromSigma-Aldrich (St. Louis, Mo.) and were used without furtherpurification unless noted otherwise.

Cell Culture.

A549, HEK293, and HepG2 cells were maintained in Dulbecco's modifiedEagle medium (DMEM) supplemented with 10% FBS in a humidified incubatorat 37° C. with 5% CO₂.

Protein Expression, Purification and Labeling.

The gene coding for the catalytic domain of PTP1B (amino acids 1-321)was amplified by the polymerase chain reaction using PTP1B cDNA astemplate and oligonucleotides5′-ggaattccatatggagatggaaaaggagttcgagcag-3′ and5′-gggatccgtcgacattgtgtggctccaggattcgtttgg-3′ as primers. The resultingDNA fragment was digested with endonucleases Nde I and Sal I andinserted into prokaryotic vector pET-22b(+)-ybbR (Yin, J et al. Proc.Natl. Acad. Sci. U.S.A. 2005, 102, 15815-15820). This cloning procedureresulted in the addition of a ybbR tag (VLDSLEFIASKL) to the N-terminusof PTP1B. Expression and purification of the ybbR-tagged PTP1B werecarried out as previously described (Ren, L et al. Biochemistry 2011,50, 2339-2356). Texas Red labeling of PTP1B was carried out by treatingthe ybbR-tagged PTP1B protein (80 μM) in 50 mM HEPES, pH 7.4, 10 mMMgCl₂ with Sfp phosphopantetheinyl transferase (1 μM) and Texas Red-CoA(100 μM) for 30 min at room temperature (Yin, J et al. Proc. Natl. Acad.Sci. U.S.A. 2005, 102, 15815-15820). The reaction mixture was passedthrough a G-25 fast-desalting column equilibrated in 30 mM HEPES, pH7.4, 150 mM NaCl to remove any free dye molecules. The full-length humanS16A/Y23A mutant Pin1 was expressed and purified from E. coli aspreviously described (Liu, T et al. J. Med. Chem. 2010, 53, 2494-2501).

Library Synthesis.

The cyclic peptide library was synthesized on 1.35 g ofaminomethyl-ChemMatrix resin (0.57 mmol/g). The library synthesis wasperformed at room temperature unless otherwise noted. The linkersequence (BBM) was synthesized using standard Fmoc chemistry. Thetypical coupling reaction contained 5 equiv of Fmoc-amino acid, 5 equivof HBTU and 10 equiv of diisopropylethylamine (DIPEA) and was allowed toproceed with mixing for 2 h. The Fmoc group was removed by treatmenttwice with 20% (v/v) piperidine in DMF (5+15 min), and the beads wereexhaustively washed with DMF (6×). To spatially segregate the beads intoouter and inner layers, the resin (after removal of N-terminal Fmocgroup) was washed with DMF and water, and soaked in water overnight. Theresin was quickly drained and suspended in a solution ofFmoc-Glu(δ-NHS)-OAll (0.10 equiv), Boc-Met-OSu (0.4 equiv) andN-methylmorpholine (2 equiv) in 20 mL of 1:1 (v/v) DCM/diethyl ether(Joo, S H et al. J. Am. Chem. Soc. 2006, 128, 13000-13009). The mixturewas incubated on a carousel shaker for 30 min. The beads were washedwith 1:1 DCM/diethyl ether (3×) and DMF (8×). Next, the Fmoc group wasremoved by piperidine treatment. Then, Fmoc-Arg(Pbf)-OH (4×),Fmoc-Nal-OH, and Fmoc-Phe-OH were sequentially coupled by standard Fmocchemistry to half of the resin. The other half was coupled with the sameamino acids in the reverse sequence. The resin was combined and therandom sequence was synthesized by the split-and-pool method using 5equiv of Fmoc-amino acids, 5 equiv HATU and 10 equiv DIPEA as thecoupling agent. The coupling reaction was repeated once to ensurecomplete coupling at each step. For random positions, a 24-amino acidset was selected based on their structural diversity, metabolicstability, and commercial availability, including 10 proteinogenicα-L-amino acids (Ala, Asp, Gln, Gly, His, Ile, Ser, Trp, Pro, and Tyr),5 nonproteinogenic α-L-amino acids (L-4-fluorophenylalanine (Fpa),L-homoproline (Pip), L-norleucine (Nle), L-phenylglycine (Phg) andL-4-(phosphonodifluoromethyl)phenylalanine (F₂Pmp)), and nine α-D-aminoacids (D-2-naphthylalanine (D-Nal), D-Ala, D-Asn, D-Glu, D-Leu, D-Phe,D-Pro, D-Thr, and D-Val). To differentiate isobaric amino acids duringPED-MS analysis, 4% (mol/mol) of CD₃CO₂D was added to the couplingreactions of D-Ala, D-Leu, and D-Pro, while 4% CH₃CD₂CO₂D was added tothe Nle reactions. Fmoc-F2Pmp-OH (0.06 equiv) and Fmoc-Tyr-OH (0.54equiv) was placed in the middle of the random positions usingHATU/DIPEA. After the entire sequence was synthesized, the allyl groupon the C-terminal Glu residue was removed by treatment with a DCMsolution containing tetrakis(triphenylphosphine)palladium [Pd(PPh₃)₄,0.25 equiv] and phenylsilane (5 equiv) for 15 min (3×). The beads weresequentially washed with 0.5% (v/v) DIPEA in DMF, 0.5% (w/v) sodiumdimethyldithiocarbamate hydrate in DMF, DMF (3×), DCM (3×), and DMF(3×). The Fmoc group on the N-terminal random residue was removed bypiperidine as described above. The beads were washed with DMF (6×), DCM(3×), and 1 M HOBt in DMF (3×). For peptide cyclization, a solution ofPyBOP/HOBt/DIPEA (5, 5, 10 equiv, respectively) in DMF was mixed withthe resin and the mixture was incubated on a carousel shaker for 3 h.The resin was washed with DMF (3×) and DCM (3×) and dried under vacuumfor >1 h. Side-chain deprotection was carried out with a modifiedreagent K 78.5:7.5:5:5:2.5:1:1 (v/v)TFA/phenol/water/thioanisole/ethanedithiol/anisole/triisopropylsilane)for 3 h. The resin was washed with TFA and DCM and dried under vacuumbefore storage at ˜20° C.

Library Screening and Peptide Sequencing.

Library resin (100 mg, 300,000 beads) was swollen in DCM, washedextensively with DMF, doubly distilled H₂O, and incubated in 1 mL ofblocking buffer (PBS, pH 7.4, 150 mM NaCl, 0.05% Tween 20 and 0.1%gelatin) containing 20 nM Texas red-labeled PTP1B at 4° C. for 3 h. Thebeads were examined under an Olympus SZX12 microscope equipped with afluorescence illuminator (Olympus America, Center Valley, Pa.) and themost intensely fluorescent beads were manually collected as positivehits. Beads containing encoding linear peptides were individuallysequenced by partial Edman degradation-mass spectrometry (PED-MS) (Liu,T et al. J. Med. Chem. 2010, 53, 2494-2501).

Individual Peptide Synthesis and Labeling.

Monocyclic and bicyclic peptides were synthesized on Rink Resin LS (0.2mmol/g) using standard Fmoc chemistry. For monocyclic peptides, afterthe last (N-terminal) residue was coupled, the allyl group on theC-terminal Glu residue was removed by treatment with Pd(PPh₃)₄ andphenylsilane (0.1 and 10 equiv, respectively) in anhydrous DCM (3×15min). The N-terminal Fmoc group was removed by treatment with 20% (v/v)piperidine in DMF and the peptide was cyclized by treatment withPyBOP/HOBt/DIPEA (5, 5, and 10 equiv) in DMF for 3 h. For bicyclicpeptides, the N-terminal Fmoc group was removed with piperidine and atrimesic acid was coupled on the N-terminal amine using HBTU as acoupling agent. The allyloxycarbonyl groups on the side chains of twoDap residues were removed by treatment with Pd(PPh₃)₄ and phenylsilane(0.1 and 10 equiv, respectively) in anhydrous DCM for 2 h. The resultingpeptide was cyclized with PyBOP as described above. The peptides weredeprotected and released from the resin by treatment with 82.5:5:5:5:2.5(v/v) TFA/thioanisole/water/phenol/ethanedithiol for 2 h. The peptideswere triturated with cold ethyl ether (3×) and purified byreversed-phase HPLC on a C18 column. The authenticity of each peptidewas confirmed by MALDI-TOF mass spectrometry. Peptide labeling with FITCwas performed by dissolving the purified peptide (˜1 mg) in 300 μL of1:1:1 (vol/vol) DMSO/DMF/150 mM sodium bicarbonate (pH 8.5) and mixingwith 10 μL of FITC in DMSO (100 mg/mL). After 20 min at roomtemperature, the reaction mixture was subjected to reversed-phase HPLCon a C18 column to isolate the FITC-labeled peptide.

PTP Inhibition Assay.

PTP assays were performed in a quartz microcuvette (total volume 150μL). The reaction mixture contains 100 mM Tris-HCl, pH 7.4, 50 mM NaCl,2 mM EDTA, 1 mM TCEP, 0-1 μM of PTP inhibitor, and 500 μMpara-nitrophenyl phosphate (pNPP). The enzymatic reaction was initiatedby the addition of PTP (final concentration 15-75 nM) and monitoredcontinuously at 405 nm on a UV-VIS spectrophotometer. Initial rates werecalculated from the reaction progress curves (typically <60 s). Thehalf-maximal inhibition constant (IC₅₀) was defined as the concentrationof an inhibitor that reduced the enzyme activity to 50% and was obtainedby plotting the rates (V) against the inhibitor concentration [I] andfitting the data against the equation

$V = \frac{V_{0}}{\left( {1 + \frac{\lbrack I\rbrack}{{IC}_{50}}} \right)\;}$where V₀ is the enzymatic reaction rate in the absence of inhibitor. Theinhibition constant (K_(i)) was determined by measuring the initialrates at fixed enzyme concentration (15 nM) but varying concentrationsof pNPP (0-24 mM) and inhibitor (0-112 nM). The reaction rate (V) wasplotted against the pNPP concentration ([S]) and fitted against theequation

$\frac{1}{V} = {{K \times \frac{1}{\lbrack S\rbrack}} + \frac{1}{V_{{ma}\; x}}}$to obtain the Michaelis constant K. The K_(i) value was obtained byplotting the K values against the inhibitor concentration [I] and fittedto equation

$\frac{K}{K_{0}} = {1 + \frac{\lbrack I\rbrack}{K_{i}}}$where K₀ is the Michaelis constant in the absence of inhibitor ([I]=0).

Confocal Microscopy.

Approximately 5×10⁴ A549 cells were seeded in 35-mm glass-bottomedmicrowell dish (MatTek) containing 1 mL of media and cultured for oneday. A549 cells were gently washed with DPBS once and treated with theFITC-labeled PTP1B inhibitors (5 μM), dextran^(Rho) (1 mg mL⁻¹) ingrowth media for 2 h at 37° C. in the presence of 5% CO₂. Thepeptide-containing media was removed and the cells were washed with DPBSthree times and incubated for 10 min in 1 mL of DPBS containing 5 μMDRAQS. The cells were again washed with DPBS twice. Then the cells wereimaged on a Visitech Infinity 3 Hawk 2D-array live cell imaging confocalmicroscope (with a 60× oil immersion lens) at 37° C. in the presence of5% CO₂. Live-cell confocal microscopic imaging of HEK293 cells aftertreatment with FITC-labeled Pin1 inhibitors were similarly conducted.

Immunoblotting.

A549 cells were cultured in full growth media to reach 80% confluence.The cells were starved in serum free media for 3 h and treated withvarying concentrations of PTP1B inhibitors for 2 h, followed by 30 minincubation in media supplemented with 1 mM sodium pervanadate. Thesolutions were removed and the cells were washed with cold DPBS twice.The cells were detached and lysed in 50 mM Tris-HCl, pH 7.4, 150 mMNaCl, 1% NP-40, 10 mM sodium pyrophosphate, 5 mM iodoacetic acid, 10 mMNaF, 1 mM EDTA, 2 mM sodium pervanadate, 0.1 mg/mL phenylmethanesulfonylfluoride, 1 mM benzamidine, and 0.1 mg/mL trypsin inhibitor. After 30min incubation on ice, the cell lysate was centrifuged at 15,000 rpm for25 min in a microcentrifuge. The total cellular proteins were separatedby SDS-PAGE and transferred electrophoretically to a PVDF membrane,which was immunoblotted using anti-phosphotyrosine antibody 4G10. Thesame samples were analyzed on a separate SDS-PAGE gel and stained byCoomassie brilliant blue to ascertain equal sample loading in all lanes.

To test the inhibitor's effect on insulin signaling pathway, HepG2 cellswere cultured to reach 80% confluence. The cells were starved for 4 h inserum free DMEM media before being treated with PTP1B inhibitor (2 h),followed by stimulation with 100 nM insulin for 5 min. The samples wereanalyzed by SDS-PAGE as described above and immunoblotted usinganti-phospho-IR/IGF1R antibody. The PVDF membrane was also probed byanti-β-actin antibody as the loading control.

Serum Stability Test.

The stability tests were carried by modifying a previously reportedprocedure (Nguyen, L T et al. PLoS One 2010, 5, e12684). Diluted humanserum (25%) was centrifuged at 15,000 rpm for 10 min, and thesupernatant was collected. A peptide stock solution was diluted into thesupernatant to a final concentration of 5 μM and incubated at 37° C. Atvarious time points (0-24 h), 200-μL aliquots were withdrawn and mixedwith 50 μL of 15% trichloroacetic acid and incubated at 4° C. overnight.The final mixture was centrifuged at 15,000 rpm for 10 min in amicrocentrifuge, and the supernatant was analyzed by reversed-phase HPLCequipped with a C18 column. The amount of remaining peptide (%) wasdetermined by integrating the area underneath the peptide peak(monitored at 214 nm) and comparing with that of the control reaction(no serum).

Fluorescence Anisotropy.

FA experiments were carried out by incubating 100 nM FITC-labeledpeptide with varying concentrations of protein in 20 mM HEPES (pH 7.4),150 mM NaCl, 2 mM magnesium acetate, and 0.1% bovine serum albumin (BSA)for 2 h at room temperature. The FA values were measured on a MolecularDevices Spectramax M5 plate reader, with excitation and emissionwavelengths at 485 and 525 nm, respectively. Equilibrium dissociationconstants (K_(D)) were determined by plotting the FA values as afunction of protein concentration and fitting the curve to the followingequation:

$Y = \frac{\left( {A_{m\; i\; n} + {\left( {{A_{{ma}\; x} \times \frac{Q_{b}}{Q_{f}}} - A_{m\; i\; n}} \right)\left( \frac{\begin{matrix}{\left( {L + x + K_{D}} \right) -} \\\sqrt{\left( {\left( {L + x + K_{D}} \right)^{2} - {4{Lx}}} \right)}\end{matrix}}{2L} \right)}} \right)}{\left( {1 + {\left( {\frac{Q_{b}}{Q_{f}} - 1} \right)\left( \frac{\left( {L + x + K_{D}} \right) - \sqrt{\left( {\left( {L + x + K_{D}} \right)^{2} - {4{Lx}}} \right)}}{2L} \right)}} \right)}$where Y is the FA value at a given protein concentration x, L is thepeptide concentration, Q_(b)/Q_(f) is the correction factor forfluorophore-protein interaction, A_(max) is the maximum FA value whenall of the peptides are bound to protein, while A_(min) is the minimumFA value when all of the peptides are free. FA competition assay wasperformed by incubating 100 nM FITC-labeled Pin1 inhibitor 5 with 1 μMPin1, followed by the addition of 0-5 μM unlabeled inhibitor. The FAvalues were measured similarly on a pate reader. IC₅₀ values wereobtained by plotting the FA values against the competitor concentrationand curve fitting using the four-parameter dose-response inhibitionequation (Prism 6, GraphPad).

A class of cell-penetrating peptides (CPPs),cyclo(Phe-Nal-Arg-Arg-Arg-Arg-Gln) (cFΦR4, where 0 or Nal isL-naphthylalanine), were recently discovered (Qian, Z et al. ACS Chem.Biol. 2013, 8, 423-431). Unlike previous CPPs, which are typicallylinear peptides and predominantly entrapped in the endosome, cFΦR4 canefficiently escape from the endosome into the cytoplasm. Short peptidecargos (1-7 aa) could be delivered into mammalian cells by directlyincorporating them into the cFΦR₄ ring. The possibility of developingbifunctional cyclic peptides containing both cell-penetrating andtarget-binding sequences as cell-permeable inhibitors againstintracellular proteins was examined. To generate specific inhibitorsagainst PTP1B, a one-bead-two-compound library was synthesized onspatially segregated ChemMatrix resin (Liu, R et al. J. Am. Chem. Soc.2002, 124, 7678-7680), in which each bead displayed a bifunctionalcyclic peptide on its surface and contained the corresponding linearpeptide in its interior as an encoding tag (FIG. 23 and FIG. 24). Thebifunctional cyclic peptides all featured the amphipathic CPP motif FΦR₄(or its inverse sequence RRRRΦF) on one side and a random pentapeptidesequence (X¹X²X³X⁴X⁵) on the other side, where X² represents a 9:1(mol/mol) mixture of Tyr and F2Pmp while X¹ and X³-X⁵ are any of the 24amino acids that included 10 proteinogenic L-amino acids (Ala, Asp, Gln,Gly, His, Ile, Pro, Ser, Tyr, Trp), 5 unnatural α-L-amino acids (F2Pmp,L-4-fluorophenylalanine (Fpa), L-norleucine (Nle), L-phenylglycine(Phg), L-pipecolic acid (Pip)), and 9 α-D-amino acids (D-Ala, D-Asn,D-Glu, D-Leu, L-β-naphthylalanine (D-Nal), D-Phe, D-Pro, D-Thr, andD-Val). The use of 9:1 Tyr/F2Pmp ratio at the X² position, together witha 5-fold reduction of the surface peptide loading, reduced the amount ofF2Pmp-containing peptides at the bead surface by 50-fold, increasing thestringency and minimizing nonspecific binding during library screening(Chen, X et al. J. Comb. Chem. 2009, 11, 604-611). Screening of thelibrary (theoretical diversity 6.6×10⁵) against Texas red-labeled PTP1Bresulted in 65 positive beads, which were individually sequenced bypartial Edman degradation-mass spectrometry (PED-MS) (Thakkar, A et al.Anal. Chem. 2006, 78, 5935-5939) to give 42 complete sequences (Table12). Interestingly, most of the selected PTP1B inhibitors contained theinverse CPP motif (RRRRΦF).

TABLE 12 Peptide Sequences Selected fromCyclic Peptide Library against PTP1B^(a). SEQ Bead ID NO. No. Sequence136  1  Pro-Pip-Gly-F₂Pmp-Tyr-Arg 137  2  Ser-Pip-Ile-F₂Pmp-F2Pmp-Arg138  3  Ile-His-Ile-F₂Pmp-Ile-Arg 139  4  Ala-D-Ala-Ile-F₂Pmp-Pip-Arg140  5  Fpa-Ser-Pip-F₂Pmp-D-Val-Arg 141  6  Pip-D-Asn-Pro-F₂Pmp-Ala-Arg142  7  Tyr-Phg-Ala-F₂Pmp-Gly-Arg 143  8  Ala-His-Ile- F₂Pmp-D-Ala-Arg144  9  Gly-D-Asn-Gly-F₂Pmp-D-Pro-Arg 145 10 D-Phe-Gln-Pip-F₂Pmp-Ile-Arg 146 11  Ser-Pro-Gly-F₂Pmp-His-Arg 147 12 Pip-Tyr-Ile-F₂Pmp-His-Arg 148 13* Ser-D-Val-Pro-F₂Pmp-His-Arg 149 14 Ala-Ile-Pro-F₂Pmp-D-Asn-Arg 150 15  Fpa-Ser-Ile-F₂Pmp-Gln-Phe 151 16 Ala-D-Ala-Phg-F₂Pmp-D-Phe-Arg 152 17  D-Asn-D-Thr-Phg-F₂Pmp-Phg-Arg 15318* Ile-Pro-Phg-F₂Pmp-Nle-Arg 154 19  Gln-Pip-Fpa-F₂Pmp-Pip-Arg 155 20 D-Asn-Ala-Fpa-F₂Pmp-Gly-Arg 156 21  D-Asn-D-Thr-Tyr-F₂Pmp-Ala-Arg 15722  D-Glu-Ala-Phg-F₂Pmp-D-Val-Arg 158 23  Ile-D-Val-Phg-F₂Pmp-Ala-Arg159 24  Tyr-D-Thr-Phg-F₂Pmp-Ala-Arg 160 25  D-Asn-Pip-Phg-F₂Pmp-Ile-Arg161 26  Pip-D-Asn-Trp-F₂Pmp-His-Arg 162 27  Tyr-Pip-D-Val-F₂Pmp-Ile-Arg163 28  D-Asn-Ser-D-Ala-F₂Pmp-Gly-Arg 164 29*D-Thr-D-Asn-D-Val-F₂Pmp-D-Ala-Arg 165 30 D-Asn-D-Thr-D-Val-F₂Pmp-D-Thr-Arg 166 31  Ser-Ile-D-Thr-F₂Pmp-Tyr-Arg167 32  D-Asn-Fpa-D-Asn-F₂Pmp-D-Leu-Arg 168 33 Tyr-D-Asn-D-Asn-F₂Pmp-Nle-Arg 169 34  D-Asn-Tyr-D-Asn-F₂Pmp-Gly-Arg 17035  Ala-Trp-D-Asn-F₂Pmp-Ala-Arg 171 36  D-Val-D-Thr-His-F₂Pmp-Tyr-Arg172 37  Pro-Phg-His-F₂Pmp-Pip-Arg 173 38  D-Asn-Phg-His-F₂Pmp-Gly-Arg174 39  Pro-Ala-His-F₂Pmp-Gly-Arg 175 40  Ala-Tyr-His-F₂Pmp-Ile-Arg 17641  D-Asn-Pip-D-Glu-F₂Pmp-Tyr-Arg 177 42  D-Val-Ser-Ser-F₂Pmp-D-Thr-Arg^(a)Fpa, L-4-fluorophenylalanine; Pip, L-homoproline; Nle, L-norleucine;Phg, L-phenylglycine; F2Pmp, L-4-(phosphonodifluoromethyl)phenylalanine.*Sequences subjected to further analysis.

Three hit sequences(D-Thr-D-Asn-D-Val-F2Pmp-D-Ala-Arg-Arg-Arg-Arg-Nal-Phe-Gln (inhibitor1), Ser-D-Val-Pro-F2Pmp-His-Arg-Arg-Arg-Arg-Nal-Phe-Gln (inhibitor 2),and Ile-Pro-Phg-F2Pmp-Nle-Arg-Arg-Arg-Arg-Nal-Phe-Gln (inhibitor 3))were resynthesized and purified by HPLC. All three peptides arecompetitive PTP1B inhibitors (Table 13), with peptide 2 being mostpotent (K₁=54 nM) (FIG. 25). Confocal microscopic analysis of humancells treated with fluorescein isothiocyanate (FITC)-labeled inhibitor 2indicated poor cellular uptake of the peptide (FIG. 26a ). It haspreviously been shown that as the size of the cargo inserted into thecFΦR₄ ring increases, the cellular uptake efficiency of the cyclicpeptides decreases (Qian, Z et al. ACS Chem. Biol. 2013, 8, 423-431).Larger rings can be more conformationally flexible and may bind lesstightly to the cell surface receptors (e.g., membrane phospholipids)during endocytosis. The negatively charged F2Pmp may also interactintramolecularly with the FΦR₄ motif and interfere with its CPPfunction.

TABLE 13 Potency of Selected Monocyclic Peptide Inhibitors against PTP1BSEQ Monocyclic IC₅₀ ID NO Inhibitor Sequence (nM) 178 1cyclo(D-Thr-D-Asn-D- ~100 Val-F₂Pmp-D-Ala-Arg- Arg-Arg-Arg-Nal-Phe- Gln)179 2 cyclo(Ser-D-Val-Pro-  ~30 F₂Pmp-His-Arg-Arg- Arg-Arg-Nal-Phe-Gln)180 3 cyclo(Ile-Pro-Phg- ~200 F₂Pmp-Nle-Arg-Arg- Arg-Arg-Nal-Phe-Gln)

To improve the cell permeability of inhibitor 2, a bicyclic system inwhich the CPP motif is placed in one ring whereas the target-bindingsequence constitutes the other ring (FIG. 23) was explored. The bicyclicsystem keeps the CPP ring to a minimal size which, according to thepreviously observed trend (Qian, Z et al. ACS Chem. Biol. 2013, 8,423-431), can result in more efficient cellular uptake. The bicyclicsystem should be able to accommodate cargos of any size, becauseincorporation of the latter does not change the size of CPP ring and,therefore, should not affect the delivery efficiency of the cyclic CPP.The use of a rigid scaffold (e.g., trimesic acid) may also help keep theCPP and cargo motifs away from each other and minimize any mutualinterference. The smaller rings of a bicyclic peptide, compared to itsmonocyclic counterpart, can result in greater structural rigidity andimproved metabolic stability.

To convert the monocyclic PTP1B inhibitor 2 into a bicyclic peptide, theGln residue (used for attachment to the solid support and peptidecyclization) was replaced with (S)-2,3-diaminopropionic acid (Dap) and asecond Dap residue was inserted at the junction of CPP and PTP1B-bindingsequences (C-terminal to His) (FIG. 23). Synthesis of the bicycle wasaccomplished by the formation of three amide bonds between a trimesicacid and the N-terminal amine and the side chains of the two Dapresidues (FIG. 27) (Lian, W et al. J. Am. Chem. Soc. 2013, 135,11990-11995). Briefly, the linear peptide was synthesized on Rink amideresin using the standard Fmoc chemistry and N^(β)-alloxycarbonyl(Alloc)-protected Dap. After removal of the N-terminal Fmoc group, theexposed amine was acylated with trimesic acid. Removal of the Allocgroups with Pd(PPh₃)₄ followed by treatment with PyBOP afforded thedesired bicyclic structure. To facilitate labeling with fluorescentprobes, a lysine was added to the C-terminus. The bicyclic peptide(peptide 4) was deprotected by TFA and purified to homogeneity by HPLC.

Bicyclic peptide 4 can act as a competitive inhibitor of PTP1B, with aK₁ value of 37 nM (FIG. 26b ). It can be highly selective for PTP1B.When assayed against p-nitrophenyl phosphate as a substrate (500 μM),inhibitor 4 had IC₅₀ values of 30 and 500 nM for PTP1B and TCPTP,respectively (FIG. 26c and Table 14). It exhibited minimal inhibition ofany of the other PTPs tested (<10% inhibition of HePTP, SHP-1, PTPRC,PTPH1, or PTPRO at 1 μM inhibitor concentration). Inhibitor 4 hasimproved cell permeability over peptide 2, as detected by live-cellconfocal microscopy of A549 cells treated with FITC-labeled inhibitor 4(FIG. 26 a). The treated cells showed both diffuse fluorescencethroughout the cytoplasm and nucleus as well as fluorescence puncta,indicating that a fraction of the inhibitors reached the cytoplasm andnucleus while the rest was likely entrapped in the endosomes. Incubationof inhibitor 4 in human serum for 24 h at 37° C. resulted in ˜10%degradation, whereas 91% of inhibitor 2 was degraded under the samecondition (FIG. 28). Overall, inhibitor 4 compares favorably with thesmall-molecule PTP1B inhibitors reported to date (Qian, Z et al. ACSChem. Biol. 2013, 8, 423-431) with respect to potency, selectivity overthe highly similar TCPTP (17-fold), cell permeability, and stability.

TABLE 14 Selectivity of Bicyclic Inhibitor 4 against Various PTPs^(a)PTP PTP1B TCPTP HePTP PTPRC SHP1 PTPRO PTPH1 IC₅₀ 30 ± 4 500 ± 250 NA NANA NA NA (nM) ^(a)NA, no significant inhibition at 1 μM inhibitor.

Inhibitor 4 was next tested for its ability to perturb PTP1B functionduring cell signaling. Treatment of A549 cells with inhibitor 4 (0-5 μM)resulted in dose-dependent increases in the phosphotyrosine (pY) levelsof a large number of proteins, consistent with the broad substratespecificity of PTP1B (Ren, L et al. Biochemistry 2011, 50, 2339) (FIG.29a ). Analysis of the same samples by Coomassie blue staining showedsimilar amounts of proteins in all samples (FIG. 29b ), indicating thatthe increased pY levels reflected increased phosphorylation (ordecreased PTP reaction) instead of changes in the total protein levels.Remarkably, the increase in tyrosine phosphorylation was alreadyapparent at 8 nM inhibitor 4. Interestingly, further increase ininhibitor concentration beyond 1 μM reversed the effect on tyrosinephosphorylation, an observation that was also made previously by Zhangand co-workers with a different PTP1B inhibitor (Xie, L et al.Biochemistry 2003, 42, 12792-12804). To obtain further evidence that theintracellular PTP1B was inhibited by peptide 4, the pY level of insulinreceptor (IR), a well-established PTP1B substrate in vivo (Elchelby, Met al. Science 1999, 283, 1544-1548; Zabolotny, J M et al. Dev. Cell2002, 2, 489-495), was monitored by immunoblotting with specificantibodies against the pY¹¹⁶²pY¹¹⁶³ site. Again, treatment withinhibitor 4 caused dose-dependent increase in insulin receptorphosphorylation up to 1 μM inhibitor and the effect leveled off athigher concentrations (FIG. 29c,d ). Taken together, these data indicatethat bicyclic inhibitor 4 can efficiently enter mammalian cells and caninhibit PTP1B in vivo. The decreased phosphorylation at higher inhibitorconcentrations may be caused by nonspecific inhibition of other PTPs(which may in turn down regulate protein tyrosine kinases). It may alsoreflect the pleiotropic roles played by PTP1B, which can both negativelyand positively regulate the activities of different protein kinases(Lessard, L et al. Biochim. Biophys. Acta 2010, 1804, 613).

To test the generality of the bicyclic approach, it was applied todesign cell permeable inhibitors against peptidyl prolyl cis-transisomerase Pin1, a potential target for treatment of a variety of humandiseases including cancer (Lu, K P and Zhou, X Z. Nat. Rev. Mol. CellBiol. 2007, 8, 904-916), for which potent, selective, and biologicallyactive inhibitors are still lacking (More, J D and Potter, A. Bioorg.Med. Chem. Lett. 2013, 23, 4283-91). Thus, a previously reportedmonocyclic peptide (5), which is a potent inhibitor against Pin1 invitro (K_(D) 258 nM) but membrane impermeable (Liu, T et al. J. Med.Chem. 2010, 53, 2494-2501), was fused with cFΦR₄ (FIG. 30). In addition,the L-Tyr at the pThr+3 position was replaced with an Arg to improve theaqueous solubility. The resulting bicyclic peptide 6 bound Pin1 with aK_(D) value of 131 nM (Table 15 and FIG. 31). Insertion of a D-Ala atthe pThr+5 position to increase the separation between the Pin1-bindingand cell-penetrating motifs improved the inhibitor potency by ˜2-fold(K_(D)=72 nM for inhibitor 7). Inhibitor 7 competed with FITC-labeledinhibitor 5 for binding to Pin1 (FIG. 32), indicating that they both canbind to the Pin1 active site. Substitution of D-Thr for D-pThr ofinhibitor 7 reduced its potency by ˜10-fold (K_(D)=620 nM for inhibitor8, Table 16), whereas further replacement of the pipecolyl residue withD-Ala abolished Pin1 inhibitory activity (peptide 9). The bicyclicinhibitors 7-9 were cell permeable (FIG. 33). Treatment of HeLa cellswith inhibitor 7 resulted in time- and dose-dependent inhibition of cellgrowth (45% inhibition after 3-day treatment at 20 μM inhibitor 7),whereas the monocyclic inhibitor 5 and inactive peptide 9 had no effect(FIG. 34). Peptide 8 also inhibited cell growth, but to a lesser extentthan inhibitor 7.

TABLE 15 Dissociation Constants of Monocyclicand Bicyclic Peptides against Pin1 as Determined by FA Analysis SEQ Pin1K_(D) ID NO Inhibitor Sequence^(a) (nM) 181 5 cyclo(D-Ala-Sar-D- 258 ±65  pThr-Pip-Nal-Tyr- Gln)-Lys-NH₂ 182 6 bicyclo[Tm(D-Ala- 131 ± 44 Sar-D-pThr-Pip-Nal- Arg-Ala)-Dap-(Phe- Nal-Arg-Arg-Arg-Arg-Dap)]-Lys-NH₂ 183 7 bicyclo[Tm(D-Ala- 72 ± 21 Sar-D-pThr-Pip-Nal-Arg-Ala-D-Ala)-Dap- (Phe-Nal-Arg-Arg- Arg-Arg-Dap)]-Lys- NH₂ 184 8bicyclo[Tm(D-Ala- 620 ± 120 Sar-D-Thr-Pip-Nal- Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-Arg- Arg-Arg-Dap)]-Lys- NH₂ 185 9 bicyclo[Tm(D-Ala- >>6000Sar-D-Thr-D-Ala-Nal- Arg-Ala-D-Ala)-Dap- (Phe-Nal-Arg-Arg-Arg-Arg-Dap)]-Lys- NH₂ ^(a)Dap, L-2,3-diaminopropionic acid; Nal,L-P-naphthylalanine; Pip, L-pipecolic acid; Sar, sarcosine; Tm, trimesicacid. For FA analysis, all peptides were labeled at the C-terminallysine side-chain with FITC.

In conclusion, a potentially general approach to designingcell-permeable bicyclic peptides against intracellular targets wasdeveloped. These preliminary studies show that replacement of thePTP1B-binding motif with other peptide sequences of differentphysicochemical properties also resulted in their efficient deliveryinto cultured mammalian cells. The availability of a generalintracellular delivery method should greatly expand the utility ofcyclic peptides in drug discovery and biomedical research.

Example 4

Also discussed herein are the CPP sequences in Table 16. Alluptake/delivery efficiencies are in Table 17 are relative to that ofcFΦR₄ (290-1F, 100%). SUV1 are small unilamellar vesicles that mimic theneutral outer membrane of mammalian cells [45% phosphatidylcholine (PC),20% phosphatidylethanolamine (PE), 20% sphingomyelin (SM), and 15%cholesterol (CHO)]. SUV2 are small unilamellar vesicles that mimic thenegatively charged endosomal membrane of mammalian cells [50% PC, 20%PE, 10% phosphatidylinositol (PI), and 20%bis(monoacylglycerol)phosphate].

Measurements were carried out fluorescence polarization usingFITC-labeled cyclic peptides against increasing concentrations ofvesicles. Experiments were performed at pH 7.4 and 5.5 (pH inside lateendosomes).

The overall delivery efficiency appears to correlate with the CPPs'binding affinity to the endosomal membrane at pH 7.4. i.e., tighterbinding leads to higher delivery efficiency.

TABLE 16 Cyclic CPPs and their cellular uptake and membrane bindingproperties. Uptake Membrane Binding K_(D) (mM) SEQ CPP Efficiency SUV1SUV1 SUV2 SUV2 ID NO Sequence (%) pH 7.4 pH 5.5 pH 7.4 pH 5.5 290-1F 186c(FΦRRRRQ) 100 4 1.1  0.66 0.63 290-12F 187 c(FfΦRrRrQ) 681 0.8 0.0260.004 290-9F 188 c(fΦRrRrQ) 602 1.2 0.81 0.033 0.012 290-11F 189c(fΦRrRrRQ) 542 2.7 0.092 0.019 290-18F 190 c(FϕrRrRq) 205 0.75 0.040.022 290-13F 191 c(FϕrRrRQ) 200 0.68 0.28 0.04 290-6F 192 c(FΦRRRRRQ)184 2.2 0.12 0.019 290-3F 193 c(RRFRΦRQ) 163 0.22 0.4 0.26 290-7F 194c(FFΦRRRRQ) 134 1.65 0.11 0.007 290-8F 195 c(RFRFRΦRQ) 98 0.4 0.39 0.082290-5F 196 c(FΦRRRQ) 97 10.1 2.4 0.066 290-4F 197 c(FRRRRΦQ) 59 7.240.54 0.11 290-10F 198 c(rRFRΦRQ) 52 1.2 0.87 0.17 290-2F 199 c(RRΦFRRQ)47 1.95 0.69 0.025 Tat 32 too weak too weak 3.3 4.5 R₉ 35 too weak tooweak 0.47 0.03 Φ = L-naphthylalanine; ϕ = D-naphthylalanine; f =D-phenylalanine; r = D-arginine; q = D-glutamine

Example 5

Cardiomyocytes are in general difficult to transfect with DNA anddelivering proteins into them by using previous CPPs have not beensuccessful. There is therefore an unmet need for delivering therapeuticproteins into heart tissues.

The disclosed cyclic CPPs are very effective in delivering proteins intocardiomyocytes. Fluorescein isothiocyanate (FITC)-labeled cyclic CPPs[c(FΦRRRRQ)-K(FITC)-NH₂ and c(fΦRrRrQ)-K(FITC)-NH₂] were synthesized andtheir internalization into mouse ventricular cardiac myocytes was testedby treating the cells with 5 μM FITC-labeled peptide for 3 h. Afterwashing away the extracellular peptides, the internalization of CPPs wasexamined by fluorescent live-cell confocal microscopy. Both peptidesexhibited significant and predominantly diffused fluorescence throughoutthe cells, indicating efficient internalization of the CPPs into cardiacmuscle cells (FIGS. 35a and 35b ). Whether the cyclic CPPs are capableof transporting full-length proteins into cardiac muscle cells wastested. Calmodulin (with an engineered Thr5Cys), a multifunctionalcalcium-binding messenger protein, was conjugated to c(FΦRRRRQ)-C-NH₂ atthe Cys residue near N-terminus through a disulfide bond. The disulfideexchange reaction is highly specific, efficient, and reversible.Further, upon entering the cytosol of cells, the disulfide linkage isexpected to be reduced to release the native protein (FIG. 35c ). TheCPP-protein conjugate was chemically labeled on amino-groups withcyanine3, which permits visualization of the internalized calmodulin.Mouse ventricular cardiac myocytes were incubated with 6 μM of theCPP-calmodulin conjugate for 3 h, and examined by live-cell confocalmicroscopy. The intracellular fluorescence signal was present throughoutthe entire cell volume and displayed a sarcomeric pattern (FIG. 35d ),indicating the internalized calmodulin was properly integrated into thecellular machinery. These data indicate that the disclosed cyclic CPPssuch as c(FΦRRRRQ) are uniquely capable of delivering small molecules aswell as proteins (likely in their native form) into cardiomyocytes withhigh efficiency, opening the door to future therapeutic applications.

Example 6

Pin1 is a phosphorylation-dependent peptidyl-prolyl cis/trans isomerase(PPIase). It contains an N-terminal WW domain and a C-terminal catalyticdomain, both of which recognize specific phosphoserine(pSer)/phosphothreonine (pThr)-Pro motifs in their protein substrates.Through cis-trans isomerization of specific pSer/pThr-Pro bonds, Pin1regulates the levels, activities, as well as intracellular localizationof a wide variety of phosphoproteins. For example, Pin1 controls the invivo stability of cyclin D1 and cyclin E and switches c-Jun, c-Fos, andNF-κB between their inactive unstable forms and active stable forms.Isomerization by Pin1 also regulates the catalytic activity of numerouscell-cycle signaling proteins such as phosphatase CDC25C and kinaseWeel. Finally, Pin1-catalyzed conformational changes in β-catenin andNF-κB lead to subcellular translocation.

Given its critical roles in cell-cycle regulation and increasedexpression levels and activity in human cancers, Pin1 has been proposedas a potential target for the development of anticancer drugs. Pin1 isalso implicated in neural degenerative diseases such as Alzheimer'sdisease. Therefore, there have been significant interests in developingspecific inhibitors against Pin1. Small-molecule inhibitors such asJuglone, PiB, dipentamenthylene thiauram monosulfide and halogenatedphenyl-isothiazolone (TME-001) generally lack sufficient potency and/orspecificity. A number of potent peptidyl Pin1 inhibitors have beenreported and are more selective than the small-molecule inhibitors.However, peptidyl inhibitors are generally impermeable to the cellmembrane and therefore have limited utility as therapeutics or in vivoprobes. A cell-permeable bicyclic peptidyl inhibitor against Pin1, inwhich one ring (A ring) featured a Pin1-binding phosphopeptide motif[D-pThr-Pip-Nal, where Pip and Nal are (R)-piperidine-2-carboxylic acidand L-naphthylalanine, respectively] while the second ring (B ring)contained a cell-penetrating peptide, Phe-Nal-Arg-Arg-Arg-Arg is shownin FIG. 36, peptide 1. Although the bicyclic peptidyl inhibitor ispotent (K_(D)=72 nM) and active in cellular assays, its D-pThr moietymight be metabolically labile due to hydrolysis by nonspecificphosphatases. The negative charges of the phosphate group might alsoimpede the cellular entry of the inhibitor. Here a non-phosphorylatedbicyclic peptidyl inhibitor against Pin1 was prepared by screening apeptide library and hit optimization. The resulting bicyclic peptidylinhibitor is potent and selective against Pin1 in vitro, cell-permeable,and metabolically stable in biological assays.

Although removal of the phosphoryl group of peptide 1 significantlyreduced its potency against Pin1, the nonphosphorylated peptide (FIG.36, peptide 2) was still a relatively potent Pin1 inhibitor (K_(D)=0.62μM). The potency of peptide 2 might be further improved by optimizingthe sequences flanking the D-Thr-Pip-Nal motif. So a second-generationbicyclic peptide library,bicyclo[Tm-(X¹X²X³-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-Arg-Arg-Arg-Dap)]-β-Ala-β-Ala-Pra-β-Ala-Hmb-β-Ala-β-Ala-Met-resin(FIG. 35, where Tm was trimesic acid, Dap was 2,3-diaminopropionic acid,β-Ala was β-alanine, Pra was L-propargylglycine, and Hmb was4-hydroxymethyl benzoic acid), by randomizing the three N-terminalresidues of peptide 2. X¹ and X² represented any of the 27 amino acidbuilding blocks that included 12 proteinogenic L-amino acids [Arg, Asp,Gln, Gly, His, Ile, Lys, Pro, Ser, Thr, Trp, and Tyr], 5nonproteinogenic α-L-amino acids [L-4-fluorophenylalanine (Fpa),L-norleucine (Nle), L-ornithine (Orn), L-phenylglycine (Phg), andL-Nal], 6 α-D-amino acids [D-Ala, D-Asn, D-Glu, D-Leu, D-Phe, andD-Val], and 4 N^(α)-methylated L-amino acids [L-N^(α)-methylalanine(Mal), L-N^(α)-methyleucine (Mle), L-N^(α)-methylphenylalanine (Mpa),and sarcosine (Sar)], while X³ was Asp, Glu, D-Asp, D-Glu, or D-Thr.Incorporation of these nonproteinogenic amino acids was expected toincrease both the structural diversity and the proteolytic stability ofthe library peptides. The library had a theoretical diversity of 5×27×27or 3645 different bicyclic peptides, most (if not all) of which wereexpected to be cell-permeable. The library was synthesized on 500 mg ofTentaGel microbeads (130 μm, ˜7.8×10⁵ beads/g, ˜350 pmol peptides/bead).Peptide cyclization was achieved by forming three amide bonds between Tmand the N-terminal amine and the sidechain amines of the two Dapresidues.

TABLE 17 Hit Sequences from Peptide Library Screening^(a) hit X¹ X² X³ 1Pro Sar D-Asp 2 Pro Sar D-Asp 3 D-Phe Fpa D-Thr 4 His Phg D-Thr 5 MpaIle D-Glu 6 Phg His D-Glu 7 Mpa Gly D-Thr ^(a)Hits 1-3 were selectedfrom 1^(st)-round screening, whereas hits 4-7 were selected after2^(nd)-round screening.

The β-Ala provides a flexible linker, while Pra serves as a handle foron-bead labeling of the bicyclic peptides with fluorescent probesthrough click chemistry. The ester linkage of Hmb enables selectiverelease of the bicyclic peptides from the resin for solution-phasebinding analysis. Finally, the C-terminal Met allows peptide releasefrom the resin by CNBr cleavage prior to MS analysis.

The library (100 mg of resin) was screened against a S16A/Y23A mutantPin1, which has a defective WW domain. The mutant Pin1 was produced as amaltose-binding protein (MBP) fusion at the N-terminus. During the firstround of screening, Texas red-labeled MBP-Pin1 was incubated with thepeptide library and fluorescent beads were removed from the libraryunder a microscope. Three positive beads had substantially greaterfluorescence intensities than the rest of hits and were directlysubjected to peptide sequencing by partial Edman degradation massspectroscopy (PED-MS) (Table 17). The other 13 fluorescent beads weresubjected to a second round of screening, during which the bicyclicpeptide on each bead was labeled with tetramethylrhodamine (TMR) azideat the Pra residue and released from the bead by treatment with a NaOHsolution.

TABLE 18 Sequences and Pin1 Binding Affinities of Peptides Used SEQK_(D) ID NO Peptide Peptide sequence (μM) 200  1bicyclo[Tm-(D-Ala-Sar-D-pThr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-0.072 ± 0.021 Arg-Arg-Arg-Arg-Dap)]-Lys 201  2bicyclo[Tm-(D-Ala-Sar-D-Thr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-0.62 ± 0.12 Arg-Arg-Arg-Dap)]-Lys 202  3bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-0.87 ± 0.17 Arg-Arg-Arg-Dap)]-Lys 203  4bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal- 0.67 ±0.12 Arg-Arg-Arg-Arg-Dap)]-Lys 204  5bicyclo[Tm-(Mpa-Gly-D-Thr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-1.08 ± 0.12 Arg-Arg-Arg-Dap)]-Lys 205  6bicyclo[Tm-(Phg-His-D-Glu-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-1.47 ± 0.19 Arg-Arg-Arg-Dap)]-Lys 206  7bicyclo[Tm-(Mpa-Ile-D-Glu-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-1.25 ± 0.20 Arg-Arg-Arg-Dap)]-Lys 207  8bicyclo[Tm-(His-Phg-D-Thr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-1.40 ± 0.24 Arg-Arg-Arg-Dap)]-Lys 208  9bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Ala)-Dap-(Phe-Nal-Arg-Arg-Arg-2.59 ± 0.37 Arg-Dap)]-Lys 209 10bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg)-Dap-(Phe-Nal-Arg-Arg-Arg- 3.42 ±0.61 Arg-Dap)]-Lys 210 11bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg-Ala)-Dap-(Phe-Nal-Arg-Arg-0.90 ± 0.25 Arg-Arg-Dap)]-Lys 211 12bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg)-Dap-(Phe-Nal-Arg-Arg-Arg-2.36 ± 0.48 Arg-Dap)]-Lys 212 13bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Ala-β-Ala)-Dap-(Phe-Nal-Arg-2.08 ± 0.31 Arg-Arg-Arg-Dap)]-Lys 213 14bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-β-Ala-D-Ala)-Dap-(Phe-Nal-Arg-1.75 ± 0.18 Arg-Arg-Arg-Dap)]-Lys 214 15bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-β-Ala-β-Ala)-Dap-(Phe-Nal-Arg-4.83 ± 0.96 Arg-Arg-Arg-Dap)]-Lys 215 16bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Ala-D-Ala-D-Ala)-Dap-(Phe-Nal-2.49 ± 0.57 Arg-Arg-Arg-Arg-Dap)]-Lys 216 17bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Tyr-D-Ala)-Dap-(Phe-Nal-Arg-2.17 ± 0.55 Arg-Arg-Arg-Dap)]-Lys 217 18bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Val-D-Ala)-Dap-(Phe-Nal-Arg-1.75 ± 0.24 Arg-Arg-Arg-Dap)]-Lys 218 19bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Arg-D-Ala)-Dap-(Phe-Nal-Arg-0.72 ± 0.09 Arg-Arg-Arg-Dap)]-Lys 219 20bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Asp-D-Ala)-Dap-(Phe-Nal-Arg-3.19 ± 0.50 Arg-Arg-Arg-Dap)]-Lys 220 21bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg-Ser-D-Phe)-Dap-(Phe-Nal- 0.57 ±0.11 Arg-Arg-Arg-Arg-Dap)]-Lys 221 22bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Arg-D-Phe)-Dap-(Phe-Nal-Arg-0.48 ± 0.07 Arg-Arg-Arg-Dap)]-Lys 222 23bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Arg-D-Val)-Dap-(Phe-Nal-Arg-1.92 ± 0.19 Arg-Arg-Arg-Dap)]-Lys 223 24bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Arg-D-Arg)-Dap-(Phe-Nal-Arg-1.31 ± 0.10 Arg-Arg-Arg-Dap)]-Lys 224 25bicyclo[Tm-(Pro-Sar-D-Asp-Pip-Nal-Arg-Arg-D-Asp)-Dap-(Phe-Nal-Arg-4.60 ± 1.42 Arg-Arg-Arg-Dap)]-Lys 225 26bicyclo[Tm-(D-Phe-4-Fpa-D-Thr-Pip-Nal-Arg-Gly-D-Ala)-Dap-(Phe-Nal-0.74 ± 0.11 Arg-Arg-Arg-Arg-Dap)]-Lys 226 27bicyclo[Tm-(D-Phe-4-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal-0.27 ± 0.08 Arg-Arg-Arg-Arg-Dap)]-Lys 227 28bicyclo[Tm-(D-Phe-Phe-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal- 1.26 ±0.28 Arg-Arg-Arg-Arg-Dap)]-Lys 228 29bicyclo[Tm-(D-Phe-3,4-diFPhe-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-0.41 ± 0.10 Nal-Arg-Arg-Arg-Arg-Dap)]-Lys 229 30bicyclo[Tm-(D-Phe-4-ClPhe-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe- 0.78 ±0.05 Nal-Arg-Arg-Arg-Arg-Dap)]-Lys 230 31bicyclo[Tm-(D-Phe-His-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal- 1.68 ±0.17 Arg-Arg-Arg-Arg-Dap)]-Lys 231 32bicyclo[Tm-(D-Phe-4-BrPhe-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe- 1.78 ±0.42 Nal-Arg-Arg-Arg-Arg-Dap)]-Lys 232 33bicyclo[Tm-(D-Ala-4-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-1.49 ± 0.11 Arg-Arg-Arg-Arg-Dap)]-Lys 233 34bicyclo[Tm-(D-Val-4-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-1.07 ± 0.16 Arg-Arg-Arg-Arg-Dap)]-Lys 234 35bicyclo[Tm-(D-2-Fpa-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal-0.59 ± 0.05 Arg-Arg-Arg-Arg-Dap)]-Lys 235 36bicyclo[Tm-(D-3-Fpa-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal-0.39 ± 0.05 Arg-Arg-Arg-Arg-Dap)]-Lys 236 37bicyclo[Tm-(D-4-Fpa-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal-0.12 ± 0.03 Arg-Arg-Arg-Arg-Dap)]-Lys 237 38bicyclo[Tm-(D-4-CyanoPhe-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-0.35 ± 0.04 Nal-Arg-Arg-Arg-Arg-Dap)]-Lys 238 39bicyclo[Tm-(D-4-Phe-Fpa-D-Ile-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal-0.46 ± 0.17 Arg-Arg-Arg-Arg-Dap)]-Lys 239 40bicyclo[Tm-(D-4-Phe-Fpa-D-Nle-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-Nal-0.71 ± 0.12 Arg-Arg-Arg-Arg-Dap)]-Lys 240 41bicyclo[Tm-(D-4-Phe-Fpa-D-homoGlu-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Phe-0.87 ± 0.08 Nal-Arg-Arg-Arg-Arg-Dap)]-Lys 241 42bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Arg-Arg- 0.98 ±0.18 Arg-Arg-Nal-Phe-Dap)]-Lys 242 43bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Arg-Arg- 1.38 ±0.16 Nal-Phe-Arg-Arg-Dap)]-Lys 243 44bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(Arg-Nal- 0.45 ±0.05 Arg-Phe-Arg-Arg-Dap)]-Lys 244 45bicyclo[Tm-(D-Phe-Fpa-D-Thr-Pip-Nal-Arg-Ala-D-Phe)-Dap-(D-Arg-Arg-3.10 ± 0.38 D-Arg-Arg-Nal-D-Phe-Dap)]-Lys 245 46cyclo(D-Ala-Sar-D-pThr-Pip-Nal-Tyr-Gln)-Lys 0.24 ± 0.04 246 47bicyclo[Tm-(D-Ala-Sar-D-Thr-D-Ala-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal- NoArg-Arg-Arg-Arg-Dap)]-Lys binding

The released peptides were incubated with 5 μM MBP-Pin1 and the increaseof fluorescence anisotropy (FA) was measured. For bicyclic peptides thatshowed ≥50% FA increase (relative to the no-protein control), thecorresponding beads (5 beads, which still contained the linear encodingpeptides) were sequenced by PED-MS to give 4 additional completesequences (Table 1). All 7 hit sequences contained a D-amino acid at theX³ position, consistent with the previous observation that Pin1 prefersD-pThr over pThr at this position. There is a strong preference forhydrophobic especially aromatic hydrophobic residues at the X¹ position,but no obvious selectivity at the X² position.

Hit Optimization.

The 6 hit sequences (hits 1 and 2 have the same sequence) wereresynthesized with a Lys added to their C-termini, labeled withfluorescein isothiocyanate (FITC), and tested for binding to Pin1 by FA(Table 18, peptides 3-8). All six peptides bound to Pin1 with moderateaffinities (K_(D)˜1 μM), but did not improve upon peptide 2 (K_(D)=0.62μM). Peptides 3 and 4 were used for structure-activity relationshipanalysis and optimization. Either expanding or contracting the size ofthe Pin1-binding ring (A ring) decreased the binding affinity (Table 18,peptides 9-16). Replacement of the Ala residue of peptide 3 with aminoacids containing side chains of different physicochemical propertiesincluding Arg, Asp, Ser, Tyr, and Val also failed to significantlyimprove the binding affinity (Table 18, peptide 17-21). On the otherhand, modification of the D-Ala residue revealed that substitution of aD-Phe at this position increases the Pin1 inhibitory activity by ˜2-fold(K_(D)=0.48 μM for peptide 22).

Peptide 4 was subjected to similar SAR studies. As observed for peptide3, modification of the Ala residue of peptide 4 (into Gly) had littleeffect (peptide 26), but replacement of the D-Ala residue with D-Pheimproved the binding affinity to Pin1 by ˜2-fold (Table 18, K_(D)=0.27μM for peptide 27). Modifications of the Fpa residue at the X² position(e.g., replacement with other halogenated phenylalanine analogs) alldecreased the inhibitor potency (peptides 28-32). Likewise, removal ofthe aromatic side chain at the X¹ position was detrimental to Pin1binding (peptides 33 and 34). However, substitution of halogenated D-Pheanalogs improved the Pin1 binding activity (peptides 35-38). Inparticular, replacement of D-Phe with D-4-fluorophenylalanine (D-Fpa)]resulted in the most potent Pin1 inhibitor of this series (K_(D)=0.12 μMfor peptide 37) (FIGS. 36 and 37 a). Further attempts to modify theD-Thr residue or the CPP motif failed to improve the Pin1 activity(Table 18, peptides 39-45).

Biological Evaluation.

To determine whether peptide 37 binds to the catalytic site of Pin1, itsability to compete with peptide 1 for binding to Pin1 by FA analysis wasexamined. Peptide 1 had previously been shown to bind to the Pin1 activesite. As expected, peptide 37 inhibited the binding of peptide 1 to Pin1with an IC₅₀ value of 190 nM (FIG. 37b ). Next, the catalytic activityof Pin1 toward a peptide substrate, Suc-Ala-Glu-Pro-Phe-pNA, in thepresence of increasing concentrations of peptide 37 was monitored.Peptide 37 inhibited the Pin1 activity in a concentration-dependentmanner, with an IC₅₀ value of 170 nM (FIG. 37c ). These resultsdemonstrate that peptide 37 binds at (or near) the active site of Pin1.

The selectivity of peptide 37 was assessed by two different tests.First, peptide 37 was tested for binding to a panel of arbitrarilyselected proteins including bovine serum albumin (BSA), protein tyrosinephosphatases 1B, SHP1, and SHP2, the Grb2 SH2 domain, Ras, and tumornecrosis factor-a. Peptide 37 bound weakly to BSA (K_(D)˜20 μM), but notany of the other six proteins. Peptide 37 was next tested for potentialinhibition of Pin4, FKBP12, and cyclophilin A, the three other commonhuman peptidyl-prolyl cis-trans isomerases. Although Pin4 isstructurally similar to Pin1 and has partially overlapping functionswith Pin1, peptide 37 only slightly inhibited Pin4 (˜15% at 5 μMinhibitor), with an estimated IC₅₀ value of ˜34 μM (FIG. 37c ). Peptide37 had no effect on the catalytic activity of FKBP12 or cyclophilin A upto 5 μM concentration. These data suggest that peptide 37 is a highlyspecific inhibitor of Pin1.

The metabolic stability of peptide 37 was evaluated by incubating it inhuman serum for varying periods of time and analyzing the reactionmixtures by reversed-phase HPLC. The pThr-containing Pin1 inhibitor 1was used as a control. After 6 h of incubation, 97% of peptide 37remained intact, while ˜50% of bicyclic peptide 1 was degraded after 3 h(FIG. 37d ). Loss of peptide 1 was accompanied by the concomitantappearance of a new peak in HPLC. Mass spectrometric analysis of the newspecies identified it as the dephosphorylation product of peptide 1(peptide 2). This result is in agreement with our previous observationthat the structurally constrained bicyclic peptides are highly resistantto proteolytic degradation. The D-pThr moiety remains susceptible tohydrolysis by the nonspecific phosphatases in human serum.

The cellular uptake efficiency of peptide 37, peptide 1, and apreviously reported membrane-impermeable monocyclic Pin1 inhibitor(Table 18, peptide 46) was assessed by incubating HeLa cells with theFITC-labeled peptides (5 μM) for 2 h and quantifying the totalintracellular fluorescence by flow cytometry analysis. As expected,untreated cells and cells treated with peptide 46 showed little cellularfluorescence, having mean fluorescence intensity (MFI) values of 101 and193, respectively (FIG. 38a ). By contrast, cells treated with peptides1 and 37 gave MFI values of 2562 and 8792, respectively. Thus, peptide37 is internalized by HeLa cells ˜4-fold more efficiently thanpeptide 1. Presumably, the negative charged phosphate group of peptide 1interacted electrostatically with the positively charged CPP motif andreduced the cellular uptake efficiency of the latter.

Inhibition of Pin1 activity has previously been shown to decrease cellproliferation. The effect of peptide 37 on the growth of HeLa cells wasexamined by using the MTT cell viability assay. The membrane impermeablepeptide 46 and a cell-permeable but inactive (defective in Pin1 binding)bicyclic peptide (Table 18, peptide 47) were used as controls. Peptide37 inhibited HeLa cell growth in a concentration-dependent manner, withan IC₅₀ value of 1.0 μM (FIG. 38b ). As expected, neither peptide 46 nor47 had any effect on cell growth. A time-course study also showedsignificant growth inhibition (>60%) after a 3-day treatment with 5 μMpeptide 37, but not with peptide 46 or 47. The phosphorylated bicyclicpeptide 1 under similar testing conditions had an IC₅₀ value of 1.8 μM.

Finally, to ascertain that Pin1 is the molecular target of peptide 37 invivo, the intracellular protein level of a well-established Pin1substrate, promyeloretinoic leukemia protein (PML), was examined bywestern blot analysis. Pin1 negatively regulates the PML level in aphosphorylation-dependent manner and inhibition of Pin1 activity isexpected to stabilize PML and increase its intracellular level. Indeed,treatment of HeLa cells with peptide 37 (0.2-5 μM) resulted inconcentration-dependent increases in the PML level (FIG. 38c,d ). Theeffect was already significant at 0.2 μM inhibitor (1.8-fold increase inthe PML level) and plateaued at ˜1 μM (3.3-fold increase). Again,bicyclic peptide 47 had no effect under the same conditions, whilepeptide 1 (the positive control, at 5 μM) increased the PML level by3.1-fold.

By screening a peptide library followed by conventional medicinalchemistry approaches, the first potent, selective, metabolically stable,and cell-permeable peptidyl inhibitor against human Pin1 has beendisclosed. Its high potency and selectivity should make it a usefulchemical probe for exploring the cellular functions of Pin1.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A cyclic peptide of Formula Ia:

or a pharmaceutically acceptable salt thereof, wherein m, n and p areindependently selected from 0 and 1, AA¹, AA², AA³, AA⁴, AA⁵, AA⁶, AA⁷,AA⁸, and AA⁹ are each independently an amino acid, wherein four or moreof the amino acids are arginine, at least four arginines areconsecutive, and at least four consecutive amino acids have alternatingchirality.
 2. The cyclic peptide of claim 1, wherein the at least fourconsecutive arginines have alternating chirality.
 3. The cyclic peptideof claim 1, wherein at least one amino acid comprises a hydrophobicaromatic residue.
 4. The cyclic peptide of claim 3, wherein the at leastone amino acid comprising the hydrophobic aromatic residue is selectedfrom the group consisting of naphthylalanine, phenylalanine, tryptophan,and tyrosine.
 5. The cyclic peptide of claim 4, wherein the at least oneamino acid comprising the hydrophobic aromatic residue is selected fromthe group consisting of naphthylalanine and phenylalanine.
 6. The cyclicpeptide of claim 5, wherein the at least one amino acid comprising thehydrophobic aromatic residue is naphthylalanine.
 7. The cyclic peptideof claim 5, wherein the at least one amino acid comprising thehydrophobic aromatic residue is phenylalanine.
 8. The cyclic peptide ofclaim 1, wherein the peptide comprises naphthylalanine or phenylalanine.9. The cyclic peptide of claim 1, wherein the peptide comprisesnaphthylalanine and phenylalanine.
 10. The cyclic peptide of claim 1wherein the peptide has a cellular uptake efficiency which is at leastabout 1.5-fold higher than c(FΦRRRRQ).
 11. The cyclic peptide of claim 1having a structure of: c(FfΦRrRrQ); c(fΦRrRrQ); c(fΦRrRrRQ); c(FφrRrRq);or c(FφrRrRQ).
 12. A cyclic peptide of claim 1, wherein: AA¹ isphenylalanine; AA² is naphthylalanine; AA³ is arginine; AA⁴ is arginine;AA^(S) is arginine; and AA⁶ is arginine.
 13. The cyclic peptideaccording to claim 12, wherein: m and n are each 0; p is 1; and AA⁹ isglutamine.
 14. The cyclic peptide according to claim 12, wherein: m is0; n and p are each 1; AA⁸ is arginine; and AA⁹ is glutamine.
 15. Thecyclic peptide according to claim 12, wherein: m is 0; n and p are each1; AA⁸ is glutamine; and AA⁹ is phenylalanine.
 16. A cyclic peptidecomprising formula IIa, IIb, or IIc:

or a pharmaceutically acceptable salt thereof, wherein m, n and p areindependently selected from 0 and 1, AA¹, AA², AA³, AA⁴, AA^(S), AA⁶,AA⁷, AA⁸, and AA⁹ are each independently an amino acid, wherein three ormore of the amino acids are arginine, and at least four consecutiveamino acids have alternating chirality; and wherein the cargo moietycomprises a detectable moiety, a therapeutic moiety, a targeting moietyor a combination thereof.
 17. The cyclic peptide of claim 16, wherein atleast four of the amino acids are arginine.
 18. The cyclic peptide ofclaim 17, wherein the at least four arginines are consecutive.
 19. Thecyclic peptide of claim 18, wherein the at least four consecutivearginines have alternating chirality.
 20. The cyclic peptide of claim16, wherein at least one amino acid comprises a hydrophobic aromaticresidue.
 21. The cyclic peptide of claim 20, wherein the at least oneamino acid comprising the hydrophobic aromatic residue is selected fromthe group consisting of naphthylalanine, phenylalanine, tryptophan, andtyrosine.
 22. The cyclic peptide of claim 21, wherein the at least oneamino acid comprising the hydrophobic aromatic residue is selected fromthe group consisting of naphthylalanine and phenylalanine.
 23. Thecyclic peptide of claim 22, wherein the at least one amino acidcomprising the hydrophobic aromatic residue is naphthylalanine.
 24. Thecyclic peptide of claim 22, wherein the at least one amino acidcomprising the hydrophobic aromatic residue is phenylalanine.
 25. Thecyclic peptide of claim 16, wherein the peptide comprisesnaphthylalanine or phenylalanine.
 26. The cyclic peptide of claim 16,wherein the peptide comprises naphthylalanine and phenylalanine.
 27. Thecyclic peptide of claim 16 wherein the peptide has a cellular uptakeefficiency which is at least about 1.5-fold higher than c(FΦRRRRQ) (SEQID NO: 186).
 28. A cyclic peptide of claim 16, wherein AA¹ isphenylalanine; AA² is naphthylalanine; AA³ is arginine; AA⁴ is arginine;AA^(S) is arginine; and AA⁶ is arginine.
 29. The cyclic peptideaccording to claim 28, wherein: m and n are each 0; p is 1; and AA⁹ isglutamine.
 30. The cyclic peptide according to claim 28, wherein: m is0; n and p are each 1; AA⁸ is arginine; and AA⁹ is glutamine.
 31. Amethod for delivering a therapeutic agent to cytoplasm of a cell,comprising administering at least one cyclic peptide of claim 16,thereby delivering the therapeutic agent to the cytoplasm of the cell.32. The method of claim 31, wherein: AA¹ is phenylalanine; AA² isnaphthylalanine; AA³ is arginine; AA⁴ is arginine; AA^(S) is arginine;and AA⁶ is arginine.
 33. The method of claim 32, wherein: m and n areeach 0; p is 1; and AA⁹ is glutamine.
 34. The method of claim 32,wherein: m is 0; n and p are each 1; AA¹ is arginine; and AA⁹ isglutamine.
 35. The method of claim 32, wherein: m is 0; n and p are each1; AA⁸ is arginine; and AA⁹ is glutamine.
 36. The method of claim 31,wherein the therapeutic agent is an inhibitor against Ras, PTP1B, Pin1,Grb2, SH2, or combinations thereof.
 37. The method of claim 31, whereinthe cyclic peptide further comprises a detectable moiety, a targetingmoiety, or a combination thereof.
 38. The cyclic peptide of claim 16,wherein the cargo moiety comprises a therapeutic moiety.
 39. The cyclicpeptide of claim 16, wherein AA¹ is phenylalanine, AA² is phenylalanine;AA³ is naphthylalanine; AA⁴ is arginine; AA^(S) is arginine; and AA⁶ isarginine.
 40. The cyclic peptide according to claim 39, wherein: m is 0;n and p are each 1; AA⁸ is arginine; and AA⁹ is glutamine.